Pharmaceutical solution having anti-tumor effect-enhancing and toxicity-reducing effect, and pharmaceutical composition comprising same

ABSTRACT

The present disclosure relates to a pharmaceutical solution having an anti-tumor efficacy-enhancing and toxicity-reducing effect, and a pharmaceutical composition comprising the solution as well as a method for using them. The pharmaceutical solution uses deuterium oxide as a solvent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application ofPCT/CN2015/079686 filed May 25, 2015, which claims priority to Chineseapplication number 201410227737.7 filed May 26, 2014.

TECHNICAL FIELD

The present disclosure belongs in the field of medicine, and relates toa pharmaceutical solution having an anti-tumor efficacy-enhancing andtoxicity-reducing effect, and a pharmaceutical composition comprisingthe solution, as well as a method for using them.

BACKGROUND ART

According to a report by the World Health Organization (WHO), in 2014there were 12 million cases diagnosed with malignancy tumors, and 8.5million deaths from malignant tumors. Therefore, effective control ofmalignant tumors is a very urgent and enormous task for peoplepracticing in medicine.

At present, the main therapies against malignant tumors are surgery,radiotherapy, and chemotherapy with various anti-tumor drugs, and thesemethods have their own characteristics but also certain limitations,especially the unremarkable efficacy often shown in chemotherapy withanti-tumor drugs. For example, a commonly used anti-tumor drug5-fluorouracil (5-FU) shows an overall response rate of only 10% as asingle drug in clinical settings. And many anti-tumor drugs haveconsiderable toxicity which is proportional to their dose. In order toimprove efficacy, an increase in dosage of a drug is needed but alsoresults in an increase in toxicity caused by the drug, which poses adilemma. Therefore, to increase the efficacy of anti-tumor drugs whilereducing their toxicity is an important strategy towards improvement ofclinical efficacy of anti-tumor drugs.

Many existing anti-tumor drugs must be systemically or topicallyadministered by injection because of their physical or chemicalproperties, pharmacokinetics and pharmacodynamics. These are commonlyused as carrier solutions such as a sodium chloride physiologicalsolution and a 5% glucose solution. These solutions only serve ascarriers for anti-tumor drugs, and they neither have any anti-tumorefficacy nor can enhance the efficacy of the anti-tumor drugs.

Deuterium oxide (heavy water, D₂O, Mw: 20.03, CAS No. 7789-20-0) can beisolated from seawater. A normal body of adult human contains about 5 gdeuterium oxide. It has been demonstrated by experiments using dogs asan animal model that exogenous deuterium oxide that reaches aconcentration of 30% in the body causes a toxic effect. Based on thisexperimental result, theoretically, assuming the body weight of an adulthuman is 60 kg, deuterium oxide that reaches 18 kg in the body starts tocause toxicity. However, it is actually impossible for a human body tohave 18 kg deuterium oxide. Furthermore, the state government ofCalifornia, USA clearly indicates in the list of chemicals known to theState that deuterium oxide does not cause cancer or defect inreproductive development. Therefore, deuterium oxide should be anon-toxic substance [D. M. Czajka: Am. J. Physiol. 201(1961): 357-362;California Proposition 65]. In 1960s, Gross P. R. et al. found thatdeuterium oxide may inhibit DNA synthase in cells (Science 133(1961):1131-1133). Afterwards, Laissue et al. studied the anti-tumor effect ofdeuterium oxide using mice as a model (Cancer Res. 42 (1982) 1125-1129),and Alterman et al. studied the anti-tumor effect of drinking deuteriumoxide in animals using nude mice bearing a human tumor cells as a model(Cancer 62(1988): 462-466. Intl. Cancer. 45(1990): 475-480). In therecent 10 years, Hartmann et al. demonstrated that deuterium oxide caninhibit in vitro proliferation of pancreatic cancer cells (AnticancerRes. 25(2005): 3407-3412), and Bahk et al. demonstrated that deuteriumoxide can inhibit in vitro proliferation of bladder cancer cells (J.Ind. Eng. Chem. 13(2007):501-507).

The aforementioned studies on the anti-tumor effect of deuterium oxidefocus on in vitro cell-based experiments. It is well acknowledgedthrough research and development of new drugs that drug candidatesshowing efficacy in in vitro cell-based experiments have a chance ofonly 1 in 100,000 to still show efficacy in vivo. Therefore, based onthe experimental results using in vitro cell culture as a model, whethera drug candidate is clinically efficacious and approvable is poorlypredictable. The only two animal studies that have been carried out bothuse a simple drinking liquid of deuterium oxide, which is a hypotonicsolution. The mechanism of action is to increase in the in vivodeuterium content to combat tumors. In order to have an effective invivo therapeutic dosage, the animals need to orally take a large dose ofthe drinking liquid of deuterium oxide. Specifically, in the animalexperiments, a drinking liquid of deuterium oxide at a concentration of30% was prepared by mixing deuterium oxide with drinking water, and usedto completely replace drinking water for the animals to drink freely andarbitrarily. Several days to weeks later, the animals reached adeuteration state with a very high level of deuterium, which starts toexert an anti-tumor effect. However, such an drinking liquid ofdeuterium oxide has serious drawbacks: 1) currently most of theclinically efficacious anti-tumor therapies are a combinationalchemotherapy of multiple anti-tumor drugs, while most anti-tumor drugscannot be orally administered for various reasons; therefore, it isdifficult to use deuterium oxide in combination with many anti-tumordrugs, and in turn it is impossible to carry out an effectivecombinational chemotherapy for patients; 2) the drinking amount of watervaries between individuals, and if deuterium oxide is drunk arbitrarily,the dosage therefore cannot be quantified, and the therapeutic effectsamong different individuals cannot be compared; and under the influenceof pharmacokinetic factors like digestion, assimilation, and metabolism,deuterium oxide needs to be taken in a large amount, and is less likelyto be approved; 3) the patients need to drink a large amount drinkingliquid of deuterium oxide in a short time, which is difficult toimplement, results in poor clinical compliance, and also incurs highcost; 4) many mid-stage or late stage cancer patients have dysphagia orhave taken a gastrointestinal surgery, and cannot orally take food orwater, let alone deuterium oxide.

Moreover, about 50% of mid-stage or late stage cancer patients havetumor metastasis and recurrence at topical sites (such as thoraciccavity, peritoneal cavity, pelvic cavity, bladder cavity, rectal lumen,buccal and nasal cavity, uterine cavity, and skin), which is the directcause of death of most patients. Recently developed topical perfusionchemotherapy has become an effective therapy in prevention or treatmentof tumor metastasis and recurrence. However, in this method anti-tumordrugs are dissolved or diluted in the sodium chloride physiologicalsolution and administered by direct perfusion and lavage, and also havethe problem of unsatisfactory efficacy. In addition, the sodium chloridephysiological solution does not have any anti-tumor efficacy-enhancingaction.

Therefore, in the art there is still a demand for a pharmaceuticalcomposition having anti-tumor efficacy, especially a pharmaceuticalcomposition employing a solution that enhances the efficacy.

SUMMARY OF THE DISCLOSURE

The present disclosure is made based on the following finding of theinventor:

1) use of deuterium oxide to dissolve or dilute other anti-tumor drugsto prepare various pharmaceutical solutions, avoids oral administration,and can apply various administration routes such as intravenousinjection or instillation, intra-arterial injection, transcatheterembolization, topical (such as thoracic cavity, peritoneal cavity,pelvic cavity, bladder cavity, rectal lumen, buccal and nasal cavity,uterine cavity, and skin) perfusion and lavage and 40° C.-48° C.hyperthermic perfusion and lavage;2) direct injection of small doses of deuterium oxide changes the cellcycle of tumor cells and kills tumor cells;3) efficacy-enhancing and toxicity reducing effect is obtained bydirectly dissolving or diluting a small dose of anti-tumors drugs indeuterium oxide, and administering the solution by injection, or bytopical (such as thoracic cavity, peritoneal cavity, pelvic cavity,bladder cavity, rectal lumen, buccal and nasal cavity, uterine cavity,articular cavity, and skin) perfusion and lavage and 40° C.-48° C.hyperthermic perfusion and lavage, which can achieve the same efficacyas a large dose of the anti-tumor drugs but avoid the toxicitytherefore; the application of deuterium oxide with hyperthermiaexhibiting an unexpected anti-tumor effect of enhancing, thereby alsoimproves quality of life of the mammal having a tumor;4) drug approvability is increased: administration of deuterium oxide byinjection allows dosage quantification, which overcomes the drawback ofshort of drug approvability due to arbitrary drinking without aquantified dose;5) safety is achieved: administration of a certain dose of deuteriumoxide by direct injection, or by topical (such as thoracic cavity,peritoneal cavity, pelvic cavity, bladder cavity, rectal lumen, buccaland nasal cavity, uterine cavity, articular cavity, and skin) perfusionand lavage and 40° C.-48° C. hyperthermic perfusion and lavage is safeto mammals.

In summary, deuterium oxide per se has anti-tumor efficacy, and whenadministered in combination with various different anti-tumor drugs, itcan significantly enhance the anti-tumor efficacy in a synergisticmanner and also reduce toxicity of these anti-tumor drugs, therebyuseful for treatment of malignant tumors, which is an unexpected effect.

An aspect of the present disclosure is to provide a pharmaceuticalsolution characterized by using deuterium oxide as a solvent.

Another aspect of the present disclosure is to provide a medicament forcombinational administration, characterized in that the medicamentcomprises the pharmaceutical solution of the present disclosure, atleast one anti-tumor drug, and optionally one or more pharmaceuticallyacceptable excipients.

Another aspect of the present disclosure is to provide a pharmaceuticalcomposition, characterized in that the pharmaceutical compositioncomprises the pharmaceutical solution of the present disclosure, atleast one anti-tumor drug, and optionally one or more pharmaceuticallyacceptable excipients.

Another aspect of the present disclosure is to provide use of thepharmaceutical solution, the pharmaceutical composition, or themedicament for combinational administration according to the presentdisclosure in the manufacture of an anti-tumor agent.

Another aspect of the present disclosure is to provide a method forpreventing or treating tumors, comprising administering atherapeutically effective amount of the pharmaceutical solution, thepharmaceutical composition, or the medicament for combinationaladministration according to the present disclosure, to a mammal having atumor.

Another aspect of the present disclosure is to provide a method forpreventing or treating tumors, characterized in that the medicament forcombinational administration comprises deuterium oxide and at least oneanti-tumor drug, wherein the deuterium oxide and the anti-tumor drug maybe administered separately or concomitantly.

Another aspect of the present disclosure is to provide a method forpreventing or treating tumors, wherein the pharmaceutical solution, themedicament for combinational administration, and the pharmaceuticalcomposition are a solution for lavage and perfusion, a solution forinjection, a suspension, an emulsion, or an embolization.

Another aspect of the present disclosure is to provide a method forpreventing or treating tumors, comprising administering atherapeutically effective amount of the pharmaceutical solution, thepharmaceutical composition, or the medicament for combinationaladministration according to the present disclosure to a mammal having atumor by various administration routes, excluding oral administration,including intravenous injection or instillation, intra-arterialinjection, topical perfusion and lavage, topical hyperthermic perfusionand lavage, trascatheter embolization, topical intratumoral andperitumoral administration.

Another aspect of the present disclosure is to provide a method forpreventing or treating malignant tumors, wherein administration byperfusion and lavage is performed by perfusing and lavaging topicalsites of the mammal having a tumor, such as thoracic cavity, peritonealcavity, pelvic cavity, bladder cavity, buccal cavity, nasal cavity,enteric cavity, uterine cavity, articular cavity, and skin, with asolution for perfusion and lavage.

Another aspect of the present disclosure is to provide a method forpreventing or treating malignant tumors, wherein in administration byhyperthermic perfusion and lavage, the temperature of the solution forperfusion and lavage is 40° C. to 48° C., preferably 42° C. to 44.5° C.,and more preferably 42° C.

Another aspect of the present disclosure is to provide a method forpreventing or treating malignant tumors, wherein in administration byperfusion and lavage or by hyperthermic perfusion and lavage, the doseof the solution for perfusion and lavage are 5 to 6,000ml/administration to the mammal having a tumor.

Another aspect of the present disclosure is to provide a method forpreventing or treating malignant tumors, wherein in administration byintravenous injection, intra-arterial injection, intrathecal injection,or intratumoral and peritumoral injection, the dose of the solution forinjection is 1 ml/kg to 20 ml/kg to the mammal having a tumor.

In the method according to the present disclosure, the mammal isselected from a rodent and a human.

Another aspect of the present disclosure is to provide use of deuteriumoxide in the manufacture of a pharmaceutical solution.

The pharmaceutical composition or the medicament for combinationaladministration according to the present disclosure shows a markedanti-tumor effect and lower toxicity than current anti-tumor drugs. Thepharmaceutical composition or the medicament for combinationaladministration according to the present disclosure is useful fortreatment or prevention of malignant tumors. Deuterium oxide shows aneffect of synergistically enhancing the anti-tumor efficacy ofanti-tumor drugs, such that a small dose of anti-tumor drugs can achievethe same efficacy as a large dose of the anti-tumor drugs, therebyreducing the amount and toxicity of anti-tumor drugs to be used. Thepharmaceutical composition or the medicament for combinationaladministration according to the present disclosure is useful forpreventing or reducing invasion, metastasis, recurrence, anddrug-resistance of tumors.

DESCRIPTION OF FIGURES

FIG. 1. Effect of deuterium oxide on the cell cycle of human coloncancer HCT-166 cells.

FIG. 2. Effect of deuterium oxide on the cell cycle of human coloncancer HCT-166 cells, as measured by the FCM method.

FIG. 3. Effect of deuterium oxide on the cell cycle of human lung cancerA549 cells.

FIG. 4. Effect of deuterium oxide on the cell cycle of human lung cancerA549 cells, as measured by the FCM method.

FIG. 5. Effect of the glucose deuterium oxide solution in combinationwith 5-FU on the tumor size of xenograft tumors from human colon cancerHCT-166 cells in nude mice.

FIG. 6. Effect of the glucose deuterium oxide solution in combinationwith 5-FU on the tumor weight of xenograft tumors from human coloncancer HCT-166 cells in nude mice.

FIG. 7. Effect of the glucose deuterium oxide solution in combinationwith 5-FU on the body weight of nude mice bearing xenograft tumors fromhuman colon cancer HCT-166 cells.

FIG. 8. Effect of the sodium chloride deuterium oxide solution incombination with Gemcitabine on the tumor size of xenograft tumors fromhuman breast cancer MCF-7 cells in nude mice.

FIG. 9. Effect of the sodium chloride deuterium oxide solution incombination with Gemcitabine on the tumor weight of xenograft tumorsfrom human breast cancer MCF-7 cells in nude mice.

FIG. 10. Effect of the sodium chloride deuterium oxide solution incombination with Gemcitabine on the body weight of nude mice bearingxenograft tumors from human breast cancer MCF-7 cells.

FIG. 11. Effect of the sodium chloride deuterium oxide solution incombination with Gemcitabine on the tumor size of xenograft tumors fromhuman lung cancer A549 cells in nude mice.

FIG. 12. Effect of the sodium chloride deuterium oxide solution incombination with Gemcitabine on the tumor weight of xenograft tumorsfrom human lung cancer A549 cells in nude mice.

FIG. 13. Effect of the sodium chloride deuterium oxide solution incombination with Gemcitabine on the body weight of nude mice bearingxenograft tumors from human lung cancer A549 cells.

In the figures, both DJ and HW refer to deuterium oxide.

DETAILED DESCRIPTION

The present application relates to the following embodiments.

Embodiment 1

A pharmaceutical solution, characterized by using deuterium oxide as asolvent.

Embodiment 2

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises sodium chloride anddeuterium oxide, being a sodium chloride deuterium oxide solutioncontaining 0.1 g to 5 g sodium chloride, preferably 0.5 g to 2.5 gsodium chloride per 100 ml solution.

Embodiment 3

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises sodium chloride anddeuterium oxide, being a sodium chloride deuterium oxide solutioncontaining 0.9 g sodium chloride per 100 ml solution, which is preparedwith deuterium oxide and sodium chloride and has a pH of 4.5 to 7.0.

Embodiment 4

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises glucose and deuterium oxide,being a glucose deuterium oxide solution containing 0.1 g to 50 gglucose, preferably 5 to 10 g, most preferably 5 g glucose per 100 mlpharmaceutical solution, which is prepared with deuterium oxide andglucose and has a pH of 3.2 to 6.5.

Embodiment 5

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises glucose and sodium chlorideand deuterium oxide, being a glucose sodium deuterium oxide solutioncontaining 0.9 g sodium chloride and 5 g glucose per 100 mlpharmaceutical solution, which is prepared with deuterium oxide, sodiumchloride and glucose and has a pH of 3.5 to 5.5.

Embodiment 6

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises sodium bicarbonate anddeuterium oxide, being a sodium bicarbonate deuterium oxide solutioncontaining 1 to 10 g, preferably 5 to 10 g, most preferably 5 g sodiumbicarbonate per 100 ml pharmaceutical solution, which is prepared withdeuterium oxide and sodium bicarbonate and has a pH of 7.5 to 8.5.

Embodiment 7

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution is a Ringer's deuterium oxide solutioncontaining 0.85 g sodium chloride, 0.012 g potassium chloride and 0.024g calcium chloride (CaCl₂.2H₂O) per 100 ml pharmaceutical solution,which is prepared with deuterium oxide, sodium chloride, potassiumchloride and calcium chloride and has a pH of 4.5 to 7.5.

Embodiment 8

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises sodium hyaluronate anddeuterium oxide, being a sodium hyaluronate deuterium oxide solutioncontaining 0.04 to 3 g, preferably 0.08 to 1.4 g sodium hyaluronate,most preferably being a sodium hyaluronate deuterium oxide solutioncontaining 0.08 or 1.0 g sodium hyaluronate, 0.9 g sodium chloride,0.142 g disodium hydrogen phosphate and 0.027 g sodium dihydrogenphosphate per 100 ml pharmaceutical solution and having a pH adjusted to6.5 to 7.5.

Embodiment 9

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises hydroxyethyl starch anddeuterium oxide, being a hydroxyethyl starch deuterium oxide solutioncontaining 3 to 8 g hydroxyethyl starch, most preferably being asolution of hydroxyethyl starch and sodium chloride in deuterium oxidecontaining 6 g hydroxyethyl starch and 0.9 g sodium chloride per 100 mlpharmaceutical solution and having a pH adjusted to 6.0 to 7.0.

Embodiment 10

The pharmaceutical solution according to Embodiment 1, characterized inthat, the pharmaceutical solution comprises hydroxypropyl-β-cyclodextrin(HP-β-CD) and deuterium oxide, being a HP-β-CD deuterium oxide solutioncontaining 0.4 to 10 g HP-β-CD, most preferably being a HP-β-CD sodiumchloride deuterium oxide solution containing 10 g HP-β-CD and 0.9 gsodium chloride per 100 ml pharmaceutical solution and having a pHadjusted to 5.0 to 7.0.

Embodiment 11

The pharmaceutical solution according to Embodiments 1 to 10,characterized in that, the pharmaceutical solution is a solution forlavage and perfusion, a solution for injection, a suspension, anemulsion, or a solution for embolization.

Embodiment 12

An anti-tumor medicament for combinational administration, characterizedin that the medicament comprises the pharmaceutical solution accordingto any one of Embodiments 1 to 10, at least one anti-tumor drug, andoptionally one or more pharmaceutically acceptable excipients.

Embodiment 13

The medicament for combinational administration according to Embodiment12, wherein the anti-tumor drug is selected from anti-tumor cytotoxicdrugs and monoclonal antibody anti-tumor drugs.

Embodiment 14

The medicament for combinational administration according to Embodiment13, wherein the anti-tumor cytotoxic drugs include antimetabolites,phytogenic anti-tumor drugs, tumor antibiotics, alkylating agents,monoclonal antibodies, and platinum preparations.

Embodiment 15

The medicament for combinational administration according to Embodiment14, wherein the antimetabolites include 5-fluorouracil, gemcitabine,floxuridine, pemetrexed, raltitrexed, fludarabine, cytarabine; the tumorantibiotics include mitomycin, epirubicin, peplomycin, daunorubicin,adriamycin, pirarubicin, aclarubicin; the platinum preparations includecisplatin, oxaliplatin, carboplatin, nedaplatin; the phytogenicanti-tumor drugs include paclitaxel, paclitaxel liposomes, paclitaxelalbumin, docetaxel, etoposide, hydroxycamptothecin; the alkylatingagents include thiotepa, carmustine, nimustine, fotemustine,estramustine, cyclophosphamide, myleran.

Embodiment 16

The medicament for combinational administration according to Embodiment14, wherein the monoclonal antibody anti-tumor drugs includebevacizumab, cetuximab, trastuzumab, panitumumab, nimotuzumab,recombinant human endostatin.

Embodiment 17

An anti-tumor pharmaceutical composition, characterized in that thepharmaceutical composition comprises the pharmaceutical solutionaccording to any one of Embodiments 1 to 11, at least one anti-tumordrug, and optionally one or more pharmaceutically acceptable excipients.

Embodiment 18

The pharmaceutical composition according to Embodiment 17, wherein theanti-tumor drug is selected from anti-tumor cytotoxic drugs andmonoclonal antibody anti-tumor drugs.

Embodiment 19

The pharmaceutical composition according to Embodiment 18, wherein theanti-tumor cytotoxic drugs include antimetabolites, phytogenicanti-tumor drugs, tumor antibiotics, alkylating agents, monoclonalantibodies, and platinum preparations.

Embodiment 20

The pharmaceutical composition according to Embodiment 19, wherein theantimetabolites include 5-fluorouracil, gemcitabine, floxuridine,pemetrexed, raltitrexed, fludarabine, cytarabine; the tumor antibioticsinclude mitomycin, epirubicin, peplomycin, daunorubicin, adriamycin,pirarubicin, aclarubicin; the platinum preparations include cisplatin,oxaliplatin, carboplatin, nedaplatin; the phytogenic anti-tumor drugsinclude paclitaxel, paclitaxel liposomes, paclitaxel albumin, docetaxel,etoposide, hydroxycamptothecin; the alkylating agents include thiotepa,carmustine, nimustine, fotemustine, estramustine, cyclophosphamide,myleran.

Embodiment 21

The pharmaceutical composition according to Embodiment 20, wherein themonoclonal antibody anti-tumor drugs include bevacizumab, cetuximab,trastuzumab, panitumumab, nimotuzumab, recombinant human endostatin.

Embodiment 22

Use of the pharmaceutical solution according to any one of Embodiments 1to 11, the medicament for combinational administration according to anyone of Embodiments 12 to 16, or the pharmaceutical composition accordingto any one of Embodiments 17 to 21, in the manufacture of an anti-tumoragent.

Embodiment 23

The pharmaceutical solution according to any one of Embodiments 1 to 11,the medicament for combinational administration according to any one ofEmbodiments 12 to 16, or the pharmaceutical composition according to anyone of Embodiments 17 to 21, for use in treatment or prevention oftumors.

Embodiment 24

A method for treating or preventing tumors, comprising administering atherapeutically effective amount of the pharmaceutical solution, thepharmaceutical composition, or the medicament for combinationaladministration according to the present disclosure to a mammal having atumor by various administration routes, excluding oral administration,including intravenous injection or instillation, intra-arterialinjection, topical perfusion and lavage, topical hyperthermic perfusionand lavage, transcatheter embolization, topical intrathecal injection,and intratumoral and peritumoral injection.

Embodiment 25

In Embodiment 23 or 24, in the administration by hyperthermic perfusionand lavage, the temperature of the solution for lavage and perfusion is40° C. to 48±1° C., preferably 42° C. to 44.5±1° C., and more preferably42±1° C.

Embodiment 26

In Embodiment 25, the administration by topical perfusion and lavage orby topical hyperthermic perfusion and lavage is performed by perfusingand lavaging topical sites, such as thoracic cavity, peritoneal cavity,pelvic cavity, bladder cavity, buccal cavity, nasal cavity, entericcavity, uterine cavity, articular cavity, and skin, of the mammal with asolution for lavage and perfusion in a dose of 5 to 6,000ml/administration.

Embodiment 27

In Embodiment 24, in the administration by intravenous injection,intra-arterial injection, intrathecal injection, or intratumoral andperitumoral injection, the dose of the solution for injection is 1 ml/kgto 20 ml/kg.

Embodiment 28

A method for treating or preventing tumors, comprising administering atherapeutically effective amount of the pharmaceutical solutionaccording to any one of Embodiments 1 to 12, the medicament forcombinational administration according to any one of Embodiments 13 to17, or the pharmaceutical composition according to any one ofEmbodiments 18 to 22 to a mammal having a tumor.

Embodiment 29

Use of deuterium oxide in the manufacture of the pharmaceutical solutionaccording to any one of Embodiments 1 to 11.

Embodiment 30

The use according to Embodiment 29, wherein the pharmaceutical solutionserves as a broad-spectrum anti-tumor efficacy-enhancing agent, andsynergistically enhances the efficacy of anti-tumor drugs.

In the pharmaceutical solution or pharmaceutical composition accordingto the present disclosure, every 100 ml of the pharmaceutical solutionor pharmaceutical composition contains 1 to 99.9 g deuterium oxide,preferably 9 to 99.9 g deuterium oxide, more preferably 20 to 99.9 gdeuterium oxide, even more preferably 30 to 99.9 g deuterium oxide, evenmore preferably 40 to 99.9 g deuterium oxide, even more preferably 50 to99.9 g deuterium oxide, even more preferably 60 to 99.9 g deuteriumoxide, even more preferably 70 to 99.9 g deuterium oxide, even morepreferably 80 to 99.9 g deuterium oxide, even more preferably 90 to 99.9g deuterium oxide, and most preferably 95 to 99.9 g deuterium oxide.

Deuterium oxide according to the present disclosure is produced bydeuterium oxide plants, and is commercially available. In the deuteriumoxide the isotope abundance of deuterium is 1 to 99.9%, preferably 30 to99.9%, more preferably 50 to 99.9%, more preferably 70 to 99.9%, andmost preferably 90 to 99.9%. For example the deuterium oxide provided byCambridge Isotope Laboratories, Inc. USA, may be used, which has anisotope abundance of deuterium of 90 to 99.9% (D 90-99.9%).

The pharmaceutically excipients may be water, sodium chloride, potassiumchloride, calcium chloride, magnesium chloride, sodium lactate, sodiumacetate, sodium citrate, sodium bicarbonate, potassium dihydrogenphosphate, disodium hydrogenphosphate, sodium bicarbonate, glucose,fructose, albumin, liposomes, hyaluronic acid, and/or polyethyleneglycol.

The anti-tumor drug is selected from anti-tumor cytotoxic drugs andmonoclonal antibody anti-tumor drugs.

The anti-tumor cytotoxic drugs include antimetabolites, phytogenicanti-tumor drugs, tumor antibiotics, alkylating agents, monoclonalantibodies, and platinum preparations. The antimetabolites include5-fluorouracil, gemcitabine, floxuridine, pemetrexed, raltitrexed,fludarabine, cytarabine; preferably 5-fluorouracil, gemcitabine, andpemetrexed. The tumor antibiotics include mitomycin, epirubicin,peplomycin, daunorubicin, adriamycin, pirarubicin, aclarubicin;preferably mitomycin and epirubicin. The platinum preparations includecisplatin, oxaliplatin, carboplatin, nedaplatin; preferably cisplatinand oxaliplatin. The phytogenic anti-tumor drugs include paclitaxel,paclitaxel liposomes, paclitaxel albumin, docetaxel, etoposide,hydroxycamptothecin; preferably paclitaxel, paclitaxel liposomes,paclitaxel albumin, and docetaxel. The alkylating agents includethiotepa, carmustine, nimustine, fotemustine, estramustine,cyclophosphamide, myleran; preferably thiotepa and carmustine.

The monoclonal antibody anti-tumor drugs include bevacizumab, cetuximab,trastuzumab, panitumumab, nimotuzumab, recombinant human endostatin;preferably bevacizumab and recombinant human endostatin.

The pharmaceutical solution according to the present disclosure isprepared by standard methods for preparing solution in the field ofpharmaceutics, and satisfies relevant standards in ChinesePharmacopoeia, United States Pharmacopoeia, and European Pharmacopoeia.

When the pharmaceutical solution according to the present disclosure iscombined with anti-tumor drugs, the dissolution or dilution ratios ofanti-tumor drugs in the pharmaceutical solution prescribed in ChinesePharmacopoeia, United States Pharmacopoeia, and European Pharmacopoeiaare used.

The medicament for combinational administration according to the presentdisclosure refers to combined administration of one pharmaceuticalsolution according to the present disclosure and one or more anti-tumordrugs.

The pharmaceutical composition according to the present disclosure is acomposite formulation prepared from a pharmaceutical solution and one ormore anti-tumor drugs.

The components in the pharmaceutical solution, the pharmaceuticalcomposition, or the medicament for combinational administrationaccording to the present disclosure may be administered separately orconcomitantly.

The pharmaceutical solution, the pharmaceutical composition, or themedicament for combinational administration according to the presentdisclosure is various preparations such as a solution for perfusion andlavage, a solution for injection, a suspension, an emulsion, or asolution for embolization.

The tumor according to the present disclosure is selected from lungcancer, colorectal cancer, primary liver cancer, esophageal cancer,gastric cancer and cardiac cancer, pancreatic cancer, renal cellcarcinoma, bladder cancer, prostate cancer, head and neck cancer,nasopharyngeal cancer, cervical cancer, ovarian cancer, breast cancer,brain tumor, bone and joint sarcoma, thyroid cancer, skin cancer,malignant melanoma, malignant lymphoma, leukemia, and complications andrecurrence of various malignant tumors, such as thoracic, peritonealand/or pelvic cavity metastasis and implantation of tumor cells, andmalignant effusion in thoracic, peritoneal and/or pelvic cavity.

Lung cancer includes small cell lung cancer (SCLC), non-small cell lungcancer and the like; colorectal cancer includes early stage colorectalcancer, advanced stage colorectal cancer, and the like; primary livercancer include the hepatic cell type, hepatic duct cell type, a mixedtype, and the like; esophageal cancer includes adenocarcinoma, squamouscell carcinoma, adenosquamous carcinoma, small cell carcinoma, and thelike; gastric cancer and cardiac cancer include adenocarcinoma, squamouscell carcinoma, adenosquamous carcinoma, small cell carcinoma, malignantgastrointestinal stromal tumor, and the like; pancreatic cancer includesductal cell carcinoma, osteoclast-like giant cell carcinoma, and thelike; renal cell carcinoma includes clear cell carcinoma and papillaryrenal cell carcinoma; bladder cancer includes urothelial carcinoma,squamous cell carcinoma, adenocarcinoma, and the like; prostate cancerincludes adenocarcinoma, ductal adenocarcinoma, urothelial carcinoma,and squamous cell carcinoma; head and neck cancer includes squamous cellcarcinoma, adenocarcinoma, and adenosquamous carcinoma; nasopharyngealcarcinoma includes squamous cell carcinoma and adenocarcinoma; cervicalcancer includes squamous cell carcinoma, adenocarcinoma, and sarcoma;ovarian cancer includes ovarian epithelial cancer, germ cell tumors, andovarian sex cord-stromal tumors; breast cancer includes epithelialtumors, mesenchymal tumors, and myoepitheliomas; brain tumors includeprimary brain tumors and brain tumor metastases; bone and joint sarcomasincludes chondrosarcoma, osteosarcoma, Ewing's sarcoma, and soft tissuesarcoma; thyroid cancer includes papillary carcinoma, follicularcarcinoma, and medullary carcinoma; skin cancer includes basal cellcarcinoma and squamous cell carcinoma; malignant melanoma includessuperficial diffusive melanoma, nodular melanoma, and acral-lentiginousmelanoma; malignant lymphomas include Hodgkin's lymphoma andnon-Hodgkin's lymphoma; leukemia includes acute leukemia and chronicleukemia.

In the method according to the present disclosure, the mammal isselected from a rodent and a human.

The word “comprise” or “comprising” used herein encompasses both thecase where the substance(s) or component(s) referred to is exclusivelyincluded, and the case where other substance(s) or component(s) thatdoes not interfere with the accomplishment of the intended objective isalso included in addition to the substance or component referred to.

Unless otherwise specified, all values used herein to indicate theamount of a substance should be construed as being modified by the term“approximately”.

The present disclosure will be further described in the below examples.It is to be understood that the scope of the present disclosure is notlimited to the examples.

All cell lines used in the examples of the present disclosure arecommercially available.

Example 1

0.9 g sodium chloride was weighed out, to which 99 g deuterium oxide (D,99.8%, from Cambridge Isotope Laboratories, Inc., USA, the same below)was added to dissolve the sodium chloride under stirring. Drops of asodium hydroxide solution were added to adjust the solution pH to 7.0,and then deuterium oxide was added to a final volume of 100 ml. Thesolution was sterilized by filtration through a 0.2 μm millipore filterand sealed in a container, to obtain a sodium chloride deuterium oxidesolution containing 0.9 g sodium chloride per 100 ml pharmaceuticalsolution according to the present disclosure.

Example 2

5 g glucose was weighed out, to which 99 g deuterium oxide was added todissolve the glucose under stirring. Drops of a hydrochloric acidsolution were added to adjust the pH to 5.5, and then deuterium oxidewas added to a final volume of 100 ml. The solution was sterilized byfiltration through a 0.2 μm millipore filter and sealed in a container,to obtain a 5% glucose deuterium oxide solution containing 5 g glucoseper 100 ml pharmaceutical solution according to the present disclosure.

Example 3

10 g glucose was weighed out, to which 99 g deuterium oxide was added todissolve the glucose under stirring. Drops of a hydrochloric acidsolution were added to adjust the pH to 5.5, and then deuterium oxidewas added to a final volume of 100 ml. The solution was sterilized byfiltration through a 0.2 μm millipore filter and sealed in a container,to obtain a 10% glucose deuterium oxide solution containing 10 g glucoseper 100 ml pharmaceutical solution according to the present disclosure.

Example 4

0.9 g sodium chloride and 5 g glucose were separately weighed out, towhich 99 g deuterium oxide was added to dissolve them under stirring.Drops of a hydrochloric acid solution were added to adjust the pH to5.0, and then deuterium oxide was added to a final volume of 100 ml. Thesolution was sterilized by filtration through a 0.2 μm millipore filter,sealed in a container, to obtain a glucose sodium chloride deuteriumoxide solution containing 0.9 g sodium chloride and 5 g glucose per 100ml pharmaceutical solution according to the present disclosure.

Example 5

5 g sodium bicarbonate was separately weighed out, to which 99 gdeuterium oxide was added to dissolve it under stirring. Drops of ahydrochloric acid solution were added to adjust the pH to 8.0, and thendeuterium oxide was added to a final volume of 100 ml. The solution wassterilized by filtration through a 0.2 μm millipore filter and sealed ina container, to obtain a 5% sodium bicarbonate deuterium oxide solutioncontaining 5 g sodium bicarbonate per 100 ml pharmaceutical solutionaccording to the present disclosure.

Example 6

0.85 g sodium chloride, 0.03 g potassium chloride and 0.033 g calciumchloride (CaCl₂.2H₂O) were separately weighed out, to which 99 gdeuterium oxide was added to dissolve them under stirring. Drops of asodium hydroxide solution were added to adjust the pH to 7.0, and thendeuterium oxide was added to a final volume of 100 ml. The solution wassterilized by filtration through a 0.2 μm millipore filter, and sealedin a container, to obtain a Ringer's deuterium oxide solution accordingto the present disclosure.

Example 7

0.8 g sodium chloride, 0.04 g potassium chloride, 0.1 g glucose, 0.006 gpotassium dihydrogen phosphate, 0.00475 g disodium hydrogen phosphate,and 0.22 g sodium bicarbonate were separately weighed out, to which 99 gdeuterium oxide was added to dissolve them under stirring. Drops of asodium hydroxide solution were added to adjust the pH to 7.0, and thendeuterium oxide was added to a final volume of 100 ml. The solution wassterilized by filtration through a 0.2 μm millipore filter, and sealedin a container, to obtain an Earle's balanced salt deuterium oxidesolution according to the present disclosure.

Example 8

A method for preparing a warm (40° C. to 48° C.) 0.08% sodiumhyaluronate deuterium oxide solution. Sodium hyaluronate, (HA, CAS:9067-32-7, Formula: (C₁₄H₂O₁₁N)_(n), Mw: 10⁵ to 10⁷). The solutioncomprising 0.08 g sodium hyaluronate, 0.8 g sodium chloride, 0.142 gdisodium hydrogen phosphate (Na₂HPO₄.12H₂O) and 0.027 g sodiumdihydrogen phosphate (NaH₂PO₄) per 100 ml solution, and having a pH of6.5 to 7.5. Detailed steps of the method: 0.8 g sodium chloride, 0.142 gdisodium hydrogen phosphate (Na₂HPO₄.12H₂O) and 0.027 g sodiumdihydrogen phosphate (NaH₂PO₄) was separately weighed out, dissolved in80 g deuterium oxide to make a solution, which was boiled andsterilized, then 0.08 g sterile sodium hyaluronate was added anddissolved, the volume was metered to 100 ml with deuterium oxide, the pHwas adjusted to 6.0 to 7.0, and the solution was sterilized by 0.2 μmmillipore filtration to obtain 0.08% sterile sodium hyaluronatedeuterium oxide solution, which was filled into a container. For topicalperfusion and lavage, the solution was warmed to a temperature of 40° C.to 48° C. in a hyperthermic perfusion and lavage apparatus, mixed withvarious anti-tumor drugs, and administered by topical perfusion andlavage.

Example 9

A method for preparing a 1.0% sodium hyaluronate deuterium oxidesolution comprising 10 g sodium hyaluronate, 8 g sodium chloride, 1.42 gdisodium hydrogen phosphate (Na₂HPO₄.12H₂O) and 0.27 g sodium dihydrogenphosphate (NaH₂PO₄) per 1000 ml solution, and having a pH of 6.5 to 7.5.Detailed steps of the method: 8 g sodium chloride, 1.42 g disodiumhydrogen phosphate (Na₂HPO₄.12H₂O) and 0.27 g sodium dihydrogenphosphate (NaH₂PO₄) was separately weighed out, dissolved in 800 gdeuterium oxide to make a solution, which was boiled and sterilized byfiltration, then 10 g sterile sodium hyaluronate was added anddissolved, the volume was metered to 1000 ml with deuterium oxide, thepH was adjusted to 6.0 to 7.5, and the solution was sterilized by 0.2 μmmillipore filtration to obtain a 1.0% sterile sodium hyaluronatedeuterium oxide solution, which was filled into a container and wasready for use.

Example 10

A method for preparing a perfusion and lavage solution of 6%hydroxyethyl starch (200/0.5) deuterium oxide solution. HydroxyethylStarch (CAS: 9005-27-0, Mw: 580.5. Formula: C₂₂H₄₄O₁₇). There are 4types of hydroxyethyl starches having different molecular weights:hydroxyethyl starch (20), hydroxyethyl starch (40), hydroxyethyl starch(130/0.4), and hydroxyethyl starch (200/0.5), all of which can be madeinto a perfusion and lavage solution of hydroxyethyl starch, and amongwhich hydroxyethyl starch (200/0.5) is preferred. Detailed steps of themethod: 60 g hydroxyethyl starch (200/0.5) was weighed out, dissolved in500 g deuterium oxide to make a solution; 9 g sodium chloride wasdissolved in another 500 g deuterium oxide, then the solution ofhydroxyethyl starch (200/0.5) deuterium oxide and the solution of sodiumchloride deuterium oxide were mixed in a ratio of 1:1, the volume wasmetered to 1000 ml with deuterium oxide, the pH was adjusted to 6.0 to7.0, and the solution was sterilized by 0.2 μm millipore filtration andsealed in a container and was ready for use.

Example 11

A method for preparing a hydroxypropyl-β-cyclodextrin deuterium oxidesolution. Hydroxypropyl-β-cyclodextrin (HP-β-CD, CAS: 128446-35-5. Mw:1431-1806. Formula: (C₄₂H₇₀O₃₅)-Hn+(C₃H₇O₂)_(n)) comprising HP-β-CD as asolute and deuterium oxide as a solvent, wherein the concentration ofHP-β-CD in the solution is 0.4 to 10 wt %, preferably 10 wt %. Detailedsteps of the method: 10 g HP-β-CD was weighed out and dissolved in 100 gdeuterium oxide under stirring. The solution contained 10 g HP-β-CD per100 ml pharmaceutical solution, the pH was adjusted to 5.0 to 7.0, andthe solution was sterilized by 0.2 μm millipore filtration and sealed ina container and was ready for use.

Example 12

A method for preparing a sulfobutylether-β-cyclodextrindeuterium oxidesolution. Sulfobutylether-β-cyclodextrin (SBE-β-CD. CAS: 25167-62-8)comprising SBE-β-CD as a solute and deuterium oxide as a solvent,wherein the concentration of SBE-β-CD in the solution is 3 to 30 wt %,preferably 10 wt %. Most preferably, the solution contained 10 gSBE-β-CD per 100 ml pharmaceutical solution. Detailed steps of themethod: 10 g SBE-β-CD was weighed out and dissolved in 100 g deuteriumoxide under stirring, the pH was adjusted to 5.0 to 7.0, and thesolution was sterilized by 0.2 pin millipore filtration and sealed in acontainer and was ready for use.

Example 13

The method for warming the solution to a temperature of 40° C. to 48°C.: for a solution of or less than 50 ml, the container containing thesolution to be used was placed on a small heater or in a water bath atthe corresponding temperature for 10 to 20 min; for a solution more than50 ml, the solution was heated to a temperature of 40° C. to 48° C. in ahyperthermic perfusion and lavage apparatus (BR-TRG-1 hyperthermicperfusion intraperitoneal treatment system, manufactured by BaoruiMedical Technology Co., Ltd, Guangzhou, China) and was ready for use.

Example 14

Studies of in vitro inhibition of growth of human tumor cells bydeuterium oxide, by the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay

Cell line: all cell lines of various types shown in Table 1 wereprovided by NANJING KEYGEN BIOTECH. CO., LTD and from ATCC, and werecommercially available (the same below).

Method:

1. A bottle of cells that had been cultured for 3 to 4 days and were inthe exponential phase was taken, a suitable amount of 0.25% Trypsin-EDTAsolution was added thereto to detach the cells from the wall, and thecells were suspended in 10 ml RPMI 1640 medium containing 10% FBS.2. The cells were counted on a hemocytometer, and generally viable cellsshould be not less than 97%.3. The cell suspension was diluted with a complete medium to prepare asuspension containing 1×10⁴ cells/ml.4. To each well of a 12-well culture plate, 300 μl cell suspension wasadded. The addition of cells should be finished within 4 hours. Theplate was placed in an incubator at 37° C. and 5% CO₂ for 24 hours.5. Powdery RPMI Medium 1640 (Gibco, USA, Cat#: 31800-022) was dissolvedin deuterium oxide (provided by Cambridge Isotope Laboratories, Inc.(US)) to prepare a cell culture medium (unless particularly specified,all culture media used in the Examples hereinafter were prepared frompowdery RPMI Medium 1640 and deuterium oxide) at a concentration shownin Table 1, which was added into the cell culture plate.6. The cell culture plate was incubated for 3 days in an incubator at37° C., 5% CO₂ and 100% humidity.7. A solution of 1 mg/ml MTT was prepared in a serum-free RPMI1640medium, added to the plate at 200 μl/well, and incubated at 37° C. for 4hours, to allow MTT to be reduced to formazan.8. The supernatant was removed by pipetting, 200 μl DMSO was added todissolve formazan, and the solution was well mixed on a platform shaker.9. Absorbance of each well was measured on an ELISA reader with adetection wavelength of 570 nm and a reference wavelength of 450 nm.10. The assay was repeated one more time.

Calculation of results: Inhibition of growth of tumor cells by deuteriumoxide was calculated using tumor cells treated with sodium chloridephysiological solution as a control group. Inhibition of cell growth wasplotted against amounts of deuterium oxide to obtain a dose responsecurve, from which the half maximal inhibitory concentration (IC₅₀) ofdeuterium oxide was derived.

Inhibition of tumor cell growth (%)=(1−OD test/OD control)×100%

TABLE 1 (a-1) In vitro inhibition of growth of human tumor cells bydeuterium oxide Inhibition of growth (%) Deuterium Colon Gastric oxidecancer cell cancer cell conc. Lung cancer cell line Pancreatic cancercell line line line (v/v) A549 NCI-H460 NCI-1975 CFPAC-1 ASPC-1 PANC-1HCT-116 BGC-823 80% 92.34 98.06 87.23 91.85 85.83 79.05 94.62 74.57 70%70.88 82.33 79.65 77.94 70.14 57.29 70.42 61.90 60% 55.42 69.50 51.9361.42 52.22 50.31 66.31 37.83 50% 42.67 53.09 39.10 45.30 44.14 31.6248.43 15.99 40% 39.37 42.05 24.03 33.67 23.99 26.73 37.43 7.50 30% 27.7130.58 6.66 26.16 9.70 17.81 30.21 12.32 20% 16.19 11.60 5.60 14.84 −7.269.06 23.32 9.09 10% 18.88 9.47 −4.95 10.75 −4.88 8.43 18.58 −3.60  5%18.05 11.27 −7.44 13.42 −0.20 14.41 21.64 −4.23 2.5%  17.64 9.40 −8.2914.05 6.35 13.88 31.55 2.15 1.2%  7.71 1.15 0.84 6.82 0.64 7.37 11.812.74

TABLE 1 (a-2) In vitro inhibition of growth of human tumor cells bydeuterium oxide Inhibition of growth (%) Deuterium Cervical BreastBladder Ovarian Prostate Brain oxide cancer cancer cancer cell cancercell cancer cancer cell Osteosarcoma Leukemia conc. cell line cell lineline line cell line line cell line cell line (v/v) HeLa MCF-7 5637 SKOV3PC-3 SF767 U2-OS K562 80% 90.11 98.08 88.43 90.42 87.08 71.22 45.8493.24 70% 85.82 89.06 74.86 74.07 73.42 49.65 29.64 88.59 60% 73.3286.41 59.76 59.84 60.84 4053 23.43 70.13 50% 65.63 63.22 52.11 41.8059.42 21.23 18.21 55.51 40% 58.26 52.45 44.32 32.76 43.19 16.50 10.2339.51 30% 45.41 40.38 36.69 16.69 26.67 8.42 11.99 22.72 20% 36.12 31.6025.56 12.64 −16.46 7.66 −3.12 9.10 10% 28.87 22.47 16.15 9.65 8.81 −8.43−8.55 −7.60  5% 16.25 15.71 10.44 8.33 −2.50 −4.41 −1.64 4.23 2.5% 14.44 11.04 −3.09 7.35 4.33 3.94 4.20 6.51 1.2%  12.11 9.78 0.84 1.820.46 4.76 −2.81 −2.47

Table 1 (b) shows IC₅₀ and IC₁₀ (expressed in v/v %) of deuterium oxideagainst different types tumor cells.

TABLE 1 (b-1) IC₅₀ and IC₁₀ for in vitro inhibition of growth of humantumor cells by deuterium oxide Colon Gastric cancer cancer cell Lungcancer cell line Pancreatic cancer cell line cell line line A549NCI-H460 NCI-1975 CFPAC-1 ASPC-1 PANC-1 HCT-116 BGC-823 IC₅₀ 55.32%48.75% 62.61% 59.43% 57.00% 65.38% 51.13% 96.68% IC₁₀ 1.52% 5.60% 27.00%3.20% 11.80% 3.03% 0.25% 26.92%

TABLE 1 (b-2) IC₅₀ and IC₁₀ for in vitro inhibition of growth of humantumor cells by deuterium oxide Cervical Breast Bladder Ovarian ProstateBrain cancer cancer cancer cancer cell cancer cancer cell OsteosarcomaLeukemia cell line cell line cell line line cell line line cell linecell line HeLa MCF-7 5637 Caov-3 PC-3 SF767 U2-OS K562 IC₅₀ 68.33%61.29% 59.08% 46.83% 61.34% 91.12% 86.25% 51.72% IC₁₀  1.8% 3.28% 4.46%11.51% 9.35% 31.85% 24.99% 14.83%

It can be seen from Table 1 that deuterium oxide shows different IC₅₀and IC₁₀ against different types of tumor cells.

The results demonstrate that deuterium oxide shows a certain inhibitoryeffect on the growth of various cancer cell lines, and higher contentsof deuterium oxide show more significant inhibition of growth of tumorcells.

Example 15

In vitro inhibition of growth of human colon cancer HCT-116 cells (amoderately drug resistant strain) by deuterium oxide in combination with5-fluorouracil (5-FU)

Cell lines: human colon cancer cells HCT-116 were provided by NANJINGKEYGEN BIOTECH. CO., LTD.

Test drugs: 5-fluorouracil (5-FU), and the sodium chloride deuteriumoxide solution, prepared in Example 1.

Method: 5-fluorouracil was diluted with the sodium chloride deuteriumoxide solution according to the concentrations shown in Table 2 andadded to a cell culture plate; the in vitro inhibition of tumor cellgrowth was measured by the MTT assay in the same manner as Example 14.

The assay was repeated one more time.

Result: it was found that 5-FU showed a certain inhibitory effect ongrowth of human colon cancer HCT-116 cells (a moderately drug resistantstrain), and can significantly enhance the anti-tumor efficacy whencombined with deuterium oxide, as shown in Table 2.

TABLE 2 Inhibition of growth of human colon cancer HCT-116 cells (amoderately drug resistant strain) by deuterium oxide in combination with5-FU Inhibition Inhibition of cell growth (%) 5-FUFinal conc. of cellgrowth (%) [5-FU + 10% Deuterium (μg/ml) (5-FU alone) oxide (v/v)] 12.535.32 80.51 6.25 26.52 67.15 3.13 18.58 62.24 1.56 11.04 51.15 0.78 9.8245.36 0.39 6.98 32.94 0.20 3.24 28.82 0.10 2.30 16.45 0.05 2.55 11.140.02 2.18 10.76

The results demonstrate that the combination of deuterium oxide and 5-FUenhances the inhibitory effect on growth of drug-resistant tumor cells(a moderately drug resistant strain); and especially at a lowconcentration of 5-FU, deuterium oxide shows a more significantefficacy-enhancing effect. Therefore, deuterium oxide has a sensitizingeffect against drug resistance.

Example 16

Inhibition of invasion of human colon cancer HCT-116 cells and humanlung cancer A549 cells by deuterium oxide in combination with5-fluorouracil or gemcitabine (by the Transwell method)

Cell lines: human colon cancer HCT-116 cells and human lung cancer A549cells were provided by NANJING KEYGEN BIOTECH. CO., LTD.

Test drugs: gemcitabine (GEM), 5-fluorouracil (5-FU), and the sodiumchloride deuterium oxide solution, prepared in Example 1.

Method: Cell Culturing 1. Cell Resuscitation

1.1. Warm water at 37° C. to 40° C. was prepared, a cryogenic vial wastaken out of liquid nitrogen and immediately put into the 37° C.-40° C.warm water, followed by vigorous shaking until the cryogenic liquid wascompletely thawed;1.2. The suspension of cryogenic cells was transferred to a centrifugaltube, 5 ml culture medium was added thereto, and the cells were wellmixed in the medium by gentle pipetting;1.3. The cell suspension was centrifuged at 800 to 1,000 rpm for 5 min,and the supernatant was discarded;1.4. A complete culture medium was added to the cell pellet which waswell mixed by gentle pipetting, the cell suspension was transferred to aculture flask, and the cells were cultured by supplementing culturemedium.

Cell Invasion Assayed by Transwell

1. Test drugs were added according to different cell treatment groups:gemcitabine or 5-FU diluted in sodium chloride physiological solutiononly, or gemcitabine or 5-FU diluted in the sodium chloride deuteriumoxide solution according to the concentrations shown in Table 3 (i.e.gemcitabine+deuterium oxide, or 5-FU+deuterium oxide), was separatelyused to treat A549 and HCT116 cells, and the cells were incubated for 24hours in an incubator at 37° C., 5% CO₂;2. After incubation of cells with the test drugs for 24 hours, serum wasremoved, and the cells were incubated under starvation with anincomplete culture medium for 24 hours;3. Matrigel was placed at 4° C. in advance to thaw overnight;4. Thawed Matrigel was 1:1 diluted with the incomplete culture medium,30 μl diluted Matrigel was added to the upper compartment of Transwelland incubated at 37° C. for 120 min to gel the Matrigel;5. Cells from different groups were digested, counted, and then adjustedto a density of 1×10⁵ cells/ml with the incomplete culture medium, 100μl cell suspension was added to the upper compartment (smallcompartment) of Transwell, and 500 μl medium containing 20% FBS wasadded to the lower compartment of Transwell;6. A 24-well cell culture plate was placed in an incubator at 37° C. and5% CO₂ to culture the cells for 24 hours;7. Cell counting: cells in the Matrigel and the upper compartment werewiped off with a cotton swab, the Transwell was removed, followed byinversion and air drying, 500 μl medium containing 0.1% crystal violetwas added to the 24-well plate, the small compartment was placed in themedium to allow the membrane to be immersed in the dye, removed afterholding at 37° C. for 30 min, washed with PBS, and photographed in 3fields along the diameter (200× magnification) for cell counting.8. The assay was repeated one more time.

Results

TABLE 3 Inhibition of invasion of A549 and HCT-116 cells by deuteriumoxide Number of cells Number of cells Group (HCT-116) (A549) Control(Sodium chloride  65.0 ± 12.0 183.0 ± 12.3 physiological solution)Deuterium oxide (1.8%) 29.3 ± 5.1  74.3 ± 11.0 Deuterium oxide (3.7%)16.3 ± 3.2 54.0 ± 2.0 Deuterium oxide (7.5%)  3.7 ± 0.6 43.0 ± 4.4Deuterium oxide (15%)  3.0 ± 1.0 29.3 ± 1.5 Deuterium oxide (30%) 1.70 ±0.6 22.0 ± 1.7

In Table 3, for example, Deuterium oxide (1.8%) means that theconcentration of deuterium oxide in the cell culture medium is 1.8%(v/v), and the same applies hereinafter.

The results demonstrate that deuterium oxide alone shows inhibition ofinvasion of A549 and HCT-116 cells, and the inhibition is proportionalto its content.

TABLE 4 Inhibition of invasion of HCT-116 cells by 5-fluorouracil (5-FU)Group Number of cells Control 76.0 ± 4.6 5-FU (0.195 ug/ml) 66.0 ± 2.65-FU (0.39 ug/ml) 47.3 ± 4.0 5-FU (0.78 ug/ml) 31.3 ± 1.5 5-FU (1.56ug/ml) 23.0 ± 4.6 5-FU (3.125 ug/ml) 12.0 ± 2.0 5-FU (6.25 ug/ml) 10.7 ±1.5 5-FU (12.5 ug/ml)  6.3 ± 0.6

In Table 4, for example, 5-FU (0.195 μg/ml) means that the concentrationof 5-FU is 0.195 μg/ml in sodium chloride physiological solution as themedium.

TABLE 5 Inhibition of invasion of HCT-116 cells by 5-FU in combinationwith deuterium oxide Number of Cell line Group cells HCT-116 Control98.7 ± 3.5  Deuterium oxide(30%) + 5-FU (0.195 ug/ml) 8.7 ± 1.5Deuterium oxide(30%) + 5-FU (0.39 ug/ml) 8.3 ± 1.2 Deuteriumoxide(30%) + 5-FU (0.78 ug/ml) 6.3 ± 0.6 Deuterium oxide(30%) + 5-FU(1.56 ug/ml) 5.0 ± 1.0 Deuterium oxide(30%) + 5-FU (3.125 ug/ml) 5.7 ±0.6 Deuterium oxide(30%) + 5-FU (6.25 ug/ml) 4.3 ± 0.6 Deuteriumoxide(30%) + 5-FU (12.5 ug/ml) 3.3 ± 0.6

In Table 5, for example, Deuterium oxide (30%)+5-FU (0.195 μg/ml) meansthat in the cell culture medium the concentration of deuterium oxide is30% (v/v), and the concentration of 5-FU is 0.195 μg/ml.

The results demonstrate that the combination of 30% deuterium oxide and5-FU significantly inhibit invasion of HCT-116 cells; in particular,when the concentration of 5-FU is only 0.195 μg/ml, the combination cansignificantly inhibit invasion of HCT-116 cells.

TABLE 6 Inhibition of invasion of A549 cells by gemcitabine (GEM) GroupNumber of cells Control 178.3 ± 13.8 GEM (1.95 nM) 160.0 ± 6.6  GEM (3.9nM) 112.7 ± 3.5  GEM (7.8 nM) 59.0 ± 2.0 GEM (15.6 nM) 51.7 ± 3.1 GEM(31.2 nM) 37.3 ± 2.3 GEM (62.5 nM) 22.3 ± 0.6 GEM (125 nM) 16.0 ± 1.0GEM (250 nM)  9.7 ± 2.1

TABLE 7 Inhibition of invasion of A549 cells by GEM in combination withdeuterium oxide Cell line Group Number of cells A549 Control 161.3 ±3.8  Deuterium oxide(30%) + GEM (1.95 nM) 31.0 ± 1.0 Deuteriumoxide(30%) + GEM (3.9 nM) 23.3 ± 2.5 Deuterium oxide(30%) + GEM (7.8 nM)21.0 ± 1.0 Deuterium oxide(30%) + GEM (15.6 nM) 13.3 ± 0.6 Deuteriumoxide(30%) + GEM (31.2 nM) 10.3 ± 0.6 Deuterium oxide(30%) + GEM (62.5nM) 11.0 ± 1.0 Deuterium oxide(30%) + GEM (125 nM)  8.0 ± 1.0 Deuteriumoxide(30%) + GEM (250 nM)  6.7 ± 0.6

The results demonstrate that the combination of deuterium oxide andgemcitabine significantly inhibit invasion of A549 cells; in particular,when the concentration of gemcitabine is only 1.95 nM, the combinationcan significantly inhibit invasion of A549 cells.

The results demonstrate that deuterium oxide shows an inhibitory effecton metastasis of various tumor cells, proportional to its dose, and canbe combined with other drugs to inhibit metastasis of tumor cells.

Example 17

Effect of deuterium oxide on the cell cycle of human lung cancer A549cells and human colon cancer HCT-116 cells

Materials

Human lung cancer A549 cells and human colon cancer HCT-116 cells wereprovided by NANJING KEYGEN BIOTECH. CO., LTD., and cultured in anincubator at 37° C., 5% CO₂ and saturated humidity.

A KGA511 Cell cycle assay kit was provided by NANJING KEYGEN BIOTECH.CO., LTD.

The flow cytometer was FACS Calibur provided by Becton-Dickinson, US.

Test drugs: gemcitabine (GEM), 5-fluorouracil (5-FU), and the sodiumchloride deuterium oxide solution, prepared in Example 1.

Method: Cell Culturing 1. Cell Resuscitation

1.1. Warm water at 37° C. to 40° C. was prepared, a cryogenic vial ofcells was taken out of liquid nitrogen and immediately put into the 37°C.-40° C. warm water, followed by vigorous shaking until the cryogenicliquid was completely thawed;1.2. The suspension of cryogenic cells was transferred to a centrifugaltube, 5 ml culture medium was added thereto, and the cells were wellmixed in the medium by gentle pipetting;1.3. The cell suspension was centrifuged at 800 to 1,000 rpm for 5 min,and the supernatant was discarded;1.4. A complete culture medium was added to the cell pellet which waswell mixed by gentle pipetting, the cell suspension was transferred to aculture flask, and the cells were cultured by supplementing culturemedium.

2. Cell Subculturing

2.1. The original culture medium was removed by pipetting when the cellcoverage reached 80% to 90% in the culture bottle;2.2. A suitable amount of trypsin (0.25%) was added to digest for 1 to 2min;2.3. When all the cells became round, an equal volume of culture mediumcontaining serum was added to cease the digestion;2.4. The cells were blown by pipetting to suspend, then drawn into a 15ml centrifugal tube, and centrifuged at 1000 rpm for 5 min;2.5. The supernatant was discarded, and the cells were re-suspended in 1to 2 ml culture medium and transferred to a culture bottle for furtherculturing.

Detection of Cell Cycle by PI Single Staining

1. Cells growing in the exponential phase were digested and inoculatedto a 6-well plate; on the next day, after the cells attached to thewall, cell culture media containing corresponding test drugs were addedaccording to different groups, and a negative control group was alsoset;2. After treatment with test drugs for 72 hours, cells were digestedwith 0.25% pancreatin (EDTA-free) and collected;3. The cells were washed with PBS once (centrifuged at 2,000 rpm for 5min), and 5×10⁵ cells were collected;4. The prepared suspension of individual cells was fixed with 70% (v/v)ethanol for 2 hours (or overnight), and stored at 4° C.; beforestaining, the fixing solution was washed away with PBS (if necessary,the cell suspension was filtered once through a 200-mesh screen);5. 100 μl RNase A was added, and the cells were water-bathed at 37° C.for 30 min;6. 400 μl PI was added and well mixed for staining, and the cells werekept at 4° C. in darkness for 30 min;7. In a detection apparatus, red fluorescence with an excitationwavelength of 488 nm was recorded.

Results

TABLE 8 Effect of deuterium oxide on the cell cycle of human coloncancer HCT-116 cells(%, x ± SD, n = 6) G1 Phase S Phase G2 PhaseControl(sodium chloride 82.53 ± 1.76 9.40 ± 1.83  8.06 ± 1.89physiological solution) Deuterium oxide(30%, v/v) 89.19 ± 3.81 6.61 ±1.41  4.21 ± 0.95 5-FU(12.5 ug/mlin sodium 32.69 ± 8.66 26.07 ± 11.3241.24 ± 2.97 chloride physiological solution) Deuterium oxide(30%, 49.16± 4.47 0.30 ± 0.26 50.53 ± 4.26 v/v) + 5-FU(12.5 ug/ml)

TABLE 9 Effect of deuterium oxide on the cell cycle of human lung cancerA549 cells(%, x ± SD, n = 6) G1 Phase S Phase G2 Phase Control (sodiumchloride 80.29 ± 5.02 13.50 ± 1.33  6.21 ± 4.52 physiological solution)Deuterium oxide(30%, v/v) 86.23 ± 3.69  0.31 ± 0.28 13.47 ± 3.94 GM (250nM in sodium 73.73 ± 3.73 15.55 ± 5.51 10.71 ± 2.39 chloridephysiological solution) Deuterium oxide(30%, v/v) + 87.32 ± 1.60  0.08 ±0.01  12.6 ± 1.46 GM(250 nM)

5-FU and gemcitabine are cytotoxic metabolite anti-tumor drugs, andmainly act on tumor cells in the DNA synthesis phase, i.e. the S phase.It can be seen in the results of Tables 8 and 9 and FIGS. 1-4 thatdeuterium oxide impeded tumor cells entering the G2 phase from the Sphase; when deuterium oxide was combined with 5-FU and gemcitabine, 5-FUand gemcitabine kill the S-phase tumor cells more effectively, resultingin significant reduction in the S-phase tumor cells, and exerting asynergistic effect.

Example 18

Studying 1: Efficacy of intraperitoneal perfusion (lavage) withdeuterium oxide in combination with 5-fluorouracil on Ehrlich ascitestumor (EAC)-bearing mice.

Studying 2: Evaluate the effectiveness of different preparations andadministration routes of deuterium oxide on nude mice beard human coloncancer HCT-116 cell.

Laboratory animal: 4 to 5 week-aged male ICR mice, provided by ShanghaiSIPPR-BK Laboratory Animal Co. Ltd.

Cell lines: Ehrlich ascites tumor cells, human colon cancer HCT-116cells were provided by NANJING KEYGEN BIOTECH. CO., LTD.

Test drugs: 5-fluorouracil (5-FU), and the sodium chloride deuteriumoxide solution, prepared in Example 1.

Studying 1 Experimental Method:

1. Modeling: EAC ascitic fluid was taken and adjusted to 1×10⁷ cells/ml,which was inoculated into the peritoneal cavity of mice at 0.1 ml/mouse.2. Grouping and dosing: the animals were randomized 3 days afterinoculation, and meanwhile drugs were given to mice in each group,following the dosing scheme shown in Table 10.

TABLE 10 Grouping and dosing scheme, NS means sodium chloridephysiological solution (same below). Drugs, I.P. Groups administrationInoculation Dosing Data Group 1 NS. 0.4 ml/animal Giving NS for 3 daysfirst, Then giving 0.4 ml NS on then giving drug on day 3, each dayuntil death of the followed by inoculation animal Group 2 5-FU20 mg/kgGiving 5-FU for 3 days Then giving5-FU20 mg/kg on Days that the alone(in NS) first, then giving drug on each day for 7 successive animal hadday 3, followed by days (10 days in total), then survived wereinoculation discontinuing the drug recorded Group 3 5-FU 30 mg/kg Giving5-FU for 3 days Then giving 5-FU 30 mg/kg Days that the alone (in NS)first, then giving drug on on each day for 7 successive animal had day3, followed by days (10 days in total), then survived were inoculationdiscontinuing the drug recorded Group 4 Sodium chloride Giving Sodiumchloride Then giving0.4 ml Sodium Days that the deuterium oxidedeuterium oxide solution chloride deuterium oxide animal had solution,alone, for 3 days first, then giving solution on each day until survivedwere 0.4 ml/animal drug on day 3, followed by death of animal, withoutdrug recorded inoculation discontinuance The above 4 groups and Group 5,Group 6, Group 7below, seven groups in total: Group 5 Sodium chlorideGiving drug for 3 days first, Then giving drug on each day Days that thedeuterium oxide then giving drug on day 3, for 10 days in total, thenanimal had solution0.1 followed by inoculation discontinuing the drugsurvived were ml/animal + recorded 5-FU20 mg/kg Group 6 Sodium chlorideGiving drug for 3 days first, Then giving drug on each day Days that thedeuterium oxide then giving drug on day 3, for 10 days in total, thenanimal had solution0.2 followed by inoculation discontinuing the drugsurvived were ml/animal + recorded 5-FU20 mg/kg Group 7 Sodium chlorideGiving drug for 3 days first, Then giving drug on each day Days that thedeuterium oxide then giving drug on day 3, for 10 days in total, thenanimal had solution 0.4 followed by inoculation discontinuing the drugsurvived were ml/animal + recorded 5-FU20 mg/kg For the 4 groups below,Sodium chloride deuterium oxide solution (0.6 ml/animal)was given 3times by intraperitoneal lavage followed by intraperitonealadministration of drugs to corresponding groups Group 8 NS 0.4 ml/animalGiving NS for 3 days first, Then giving 0.4 ml NS on Days that the thengiving drug on day 3, each day until death of the animal had followed byinoculation animal survived were recorded Group 9 Sodium chloride Givingdrug for 3 days first, Then giving drug on each day Days that thedeuterium oxide then giving drug on day 3, for 10 days in total, thenanimal had solution 0.1 followed by inoculation discontinuing the drugsurvived were ml/animal + 5-FU, recorded 20 mg/kg Group 10 Sodiumchloride Giving drug for 3 days first, Then giving drug on each day Daysthat the deuterium oxide then giving drug on day 3, for 10 days intotal, then animal had solution 0.2 followed by inoculationdiscontinuing the drug survived were ml/animal + 5-FU, recorded 20 mg/kgGroup 11 Sodium chloride Giving drug for 3 days first, Then giving drugon each day Days that the deuterium oxide then giving drug on day 3, for10 days in total, then animal had solution 0.4 followed by inoculationdiscontinuing the drug survived were ml/animal + 5-FU, recorded 20 mg/kg

In Table 10, “Sodium chloride deuterium oxide solution 0.1ml/animal+5-FU 20 mg/kg” means that each mouse was given 0.1 ml sodiumchloride deuterium oxide solution, in which 5-FU had been dissolved tomake a dose of 20 mg/kg.

3. Observation of Indicators

After the dosing, ascitic fluid was taken from 3 mice from each group,the volume of the ascitic fluid was measured, the number of tumor cellsin the ascitic fluid was counted, and the rest of animals werecontinuously fed. Median survival time (MST) was recorded for animals ineach group to evaluate the survival time for each group.

The comparison between the treatment group and the control group wasexpressed by T/C (%) which is calculated by the following equation:

${{T/C}\mspace{14mu} \%} = {\frac{TMST}{CMST} \times 100\%}$

T MST: MST of the treatment group; C MST: MST of the negative controlgroup.

The evaluation criterion uses 125% as a cut-off threshold. When T/C%≧125%, effective, otherwise ineffective.

4. Statistics

The mean value was expressed in X±SD. Inter-group analysis wasstatistically performed with t test. The results were statisticallyanalyzed using SPSS (Statistical Package for the Social Science) 17.0.

5. Results 5.1. Survival Time: Effect of Intraperitoneal Administrationof Drugs on Survival Time (in Days) of Animals

TABLE 11 Effect of intraperitoneal administration of drugs on survivaltime (in days) of animals(x ± SD, n = 8) Group MST(days) T/C(%) controlgroup: NS (0.4 ml/animal) 14.1 ± 1.9  5-FU (20 mg/kg)(in NS) 21.8 ± 2.9*154.6% 5-FU (30 mg/kg)(in NS) 22.3 ± 4.9* 158.1% Sodium chloridedeuterium oxide  17.2 ± 3.2** 121.9% solution (0.4 ml/animal) Sodiumchloride deuterium oxide solution 21.4 ± 3.1* 151.7% (0.1 ml/animal) +5-FU(20 mg/kg) Sodium chloride deuterium oxide solution 24.1 ± 2.7*170.9% (0.2 ml/animal) + 5-FU(20 mg/kg) Sodium chloride deuterium oxidesolution 25.7 ± 2.6* 182.2% (0.4 ml/animal) + 5-FU(20 mg/kg) Compared tothe control group: *P < 0.001, **P < 0.05.

TABLE 12 Effect of intraperitoneal lavage with sodium chloride deuteriumoxide solution, followed by intraperitoneal administration of drugs, onsurvival time (in days) of animals(x ± SD, n = 8) Group MST(days) T/C(%)control group: NS (0.4 ml/animal) 15.3 ± 1.9  Sodium chloride deuteriumoxide solution 26.1 ± 3.7* 170.5% (0.1 ml/animal) + 5-FU (20 mg/kg)Sodium chloride deuterium oxide solution 29.1 ± 3.1* 190.1% (0.2ml/animal) + 5-FU (20 mg/kg) Sodium chloride deuterium oxide solution34.5 ± 3.3* 225.4% (0.4 ml/animal) + 5-FU (20 mg/kg) Compared to thecontrol group: *P < 0.001.

The results demonstrate that, as compared to the NS control group, thegroup receiving combined sodium chloride deuterium oxide solution, and5-FU showed an extended MST, with a P value<0.01 and T/C (%)≧150%, whichwas also better than the group receiving 5-FU alone.

Intraperitoneal lavage with the sodium chloride deuterium oxide solutionfirst, followed by intraperitoneal administration of drugs, can achievea significantly extended MST in mice, longer than the MST resulting fromonly intraperitoneal administration of drugs in the sodium chloridedeuterium oxide solution, namely 29.1±3.1 days vs. 24.1±2.7 days; and34.5±3.3 days vs. 25.7±2.6 days, both having a P value<0.05,demonstrating an advantage of intraperitoneal lavage with deuteriumoxide followed by administration of drugs.

Studying 2: Experimental Method:

1. Modeling: human colon cancer HCT-116 cells was taken and adjusted to1×107 cells/ml, which was inoculated into the peritoneal cavity of miceat 0.1 ml/mouse.2. Grouping and dosing: the animals were randomized 3 days afterinoculation, and meanwhile agents were given to mice in each group,following the dosing scheme and results shown in Table 13.

TABLE 13 Effects of the oral drinking or i.v. dosing of deuterium oxideon survival time(in days) in mice bearing human colon cancer HCT- 116cells(x ± SD, n = 6), grouping, dosing scheme and results.administration Groups agent route and dosing MST (days) T/C (%) Group 1drinking water drinking freely 36 ± 3.1  Group 2 30% deuterium drinkingfreely 39 ± 2.2  8.3 oxide, Group 3 sodium chloride 20 ml/kg, i.v. x 746 ± 2.2*  27.7 deuterium oxide solution Group 4 30% deuterium 1.deuterium oxide 53 ± 4.3** 47.2 oxide + 5-FU drinking freely 2. 5-FU 10mg/kg dissolving in saline solution, i.v. x 7 Group 5 sodium chloride5-FU 10 mg/kg 65 ± 6.2** 80.5 deuterium oxide dissolving in sodiumsolution + 5-FU chloride deuterium oxide solution, 20 ml/kg, i.v. x 7Compared to group 1: *P < 0.01, **P < 0.001; i.v. means intravenousroute.

The intravenous route of deuterium oxide show unexpected effect ofextension the survival time of mice bearing human colon cancer HCT-116cells. The drinking of 30% deuterium oxide only extended 3 days forsurvival time, P>0.05. But the intravenous administration of deuteriumoxide increased 10 days for survival time. The drinking of 30% deuteriumoxide in combination with 5-FU could extend 17 days for survival time,but the intravenous route of deuterium oxide in combination with 5-FUwould increase 30 days for survival time ultimately, in a T/C 80.5%,P<0.001. Both intravenous route of deuterium oxide were significantlysuperior to the oral drinking of administration. Conclusion: theintravenous route changes the pharmacodynamics of deuterium oxide andenhances the efficacy of it, also improves the survival time. Finally,it will be beneficial to patients with cancer therapy.

Example 19

Inhibition of liver cancer H22 ascites tumor in tumor-bearing mice by42±1° C. hyperthermic intraperitoneal retention lavage using deuteriumoxide in combination with cisplatin, and using deuterium oxide (42° C.)in combination with cisplatin.

Experimental Method:

Animal: 4 to 5 week-aged male Kunming mice, provided by ShanghaiSIPPR-BK Laboratory Animal Co. Ltd.

Cell lines: Liver cancer H22 ascites tumor cell line was provided byNANJING KEYGEN BIOTECH. CO., LTD.

Modeling: 0.2 ml suspension of H22 tumor cells containing 1×10⁷ cells/ml(2×10⁶ H22 cells in total) was inoculated into the peritoneal cavity ofmice.

Test drugs: cisplatin, and the Ringer's deuterium oxide solution asprepared in Example 6.

Grouping and dosing: the mouse model was established 7 to 8 days afterinoculation, and the mice were randomized with 18 animals/group.

Cisplatin were dissolved in a 42±1° C. Ringer's deuterium oxidesolution, and used to lavage the peritoneal cavity of the tumor-bearingmice.

1). Blank control group (normal Ringer's solution, 10 ml/kg, roomtemperature);2). Normal chemo group (cisplatin 0.6 mg/kg+normal Ringer's solution, 10ml/kg, room temperature);3). Room temperature low-dose deuterium oxide+cisplatin group (Ringer'sdeuterium oxide solution, 5 ml/kg+cisplatin 0.6 mg/kg);4). Room temperature mid-dose deuterium oxide+cisplatin group (Ringer'sdeuterium oxide solution, 20 ml/kg+cisplatin 0.6 mg/kg);5). Room temperature high-dose deuterium oxide+cisplatin group (Ringer'sdeuterium oxide solution, 40 ml/kg+cisplatin 0.6 mg/kg);6). Hyperthermic low-dose deuterium oxide+cisplatin group (42±1° C.Ringer's deuterium oxide solution, 5 ml/kg+cisplatin 0.6 mg/kg);7). Hyperthermic mid-dose deuterium oxide+cisplatin group (42±1° C.Ringer's deuterium oxide solution, 20 ml/kg+cisplatin 0.6 mg/kg);8). Hyperthermic high-dose deuterium oxide+cisplatin group (42±1° C.Ringer's deuterium oxide solution, 40 ml/kg+cisplatin 0.6 mg/kg);

The peritoneal cavity of mice was lavaged with the above compositionswhich were retained for 10 min in the peritoneal cavity after beingintroduced, which was repeated for 3 times. The lavage was performedonce another day for 5 successive times. The body weight and abdomencircumference of mice were measured on each day before administration ofthe drugs, and the daily living status of mice was observed. 24 hoursafter the administration of drugs was completed (on day 11), 8 mice fromeach group were sacrificed, the volume of ascitic fluid was measured,and bodies of the mice were dissected to observe the peritoneal organsand metastasis to lung. The rest of mice were observed for theirsurvival time, and the extension of life was calculated.

4. Observation of indicators: the same as Example 18.5. Statistics: the same as Example 18.

6. Results

TABLE 14 Effect of 42 ± 1° C. intraperitoneal lavage with deuteriumoxide in combination with cisplatin on survival time (in days) ofanimals(x ± SD, n = 10) Group MST(days) T/C(%) Blank control group(Ringer's solution for 13.2 ± 1.9  injection, 5 ml/kg) Normal chemogroup (cisplatin 0.6 mg/kg + 18.1 ± 3.2* 137.1 Ringer's solution5 ml/kg)RT low-dose deuterium oxide + cisplatin group 20.3 ± 2.2* 153.7(Ringer's deuterium oxide solution5 ml/kg + cisplatin 0.6 mg/kg) RTmid-dose deuterium oxide + cisplatin 24.7 ± 1.8* 187.1 group (Ringer'sdeuterium oxide solution20 ml/kg + cisplatin 0.6 mg/kg) RT high-dosedeuterium oxide + cisplatin group 26.1 ± 5.9* 197.7 (Ringer's deuteriumoxide solution 40ml/kg + cisplatin 0.6 mg/kg) Hyperthermic low-dosedeuterium oxide + 24.3 ± 3.3* 184.0 cisplatin group(Ringer's deuteriumoxide solution 5 ml/kg + cisplatin0.6 mg/kg) Hyperthermic mid-dosedeuterium oxide + 28.6 ± 6.4* 216.6 cisplatin group(Ringer's deuteriumoxide solution 20 ml/kg + cisplatin0.6 mg/kg) Hyperthermic high-dosedeuterium oxide + 32.1 ± 3.1* 243.1 cisplatin group(Ringer's deuteriumoxide solution40 ml/kg + cisplatin0.6 mg/kg) Compared to the negativecontrol group: *P < 0.001. RT means room temperature.

TABLE 15 Effect of 42 ± 1° C. intraperitoneal lavage with deuteriumoxide in combination with cisplatinon the volume of ascitic fluid(x ±SD, n = 8) Ascitic Groups fluid (ml) Blank control group (Ringer'ssolution for 14.85 ± 2.56  injection, 5 ml/kg) Normal chemo group(cisplatin 0.6 mg/kg + 7.30 ± 2.32* Ringer's solution for injection 5ml/kg) RT low-dose deuterium oxide + cisplatin group 6.55 ± 3.95*(Ringer's deuterium oxide solution5 ml/kg + cisplatin 0.6 mg/kg) RTmid-dose deuterium oxide + cisplatin group 4.93 ± 3.05* (Ringer'sdeuterium oxide solution20 ml/kg + cisplatin 0.6 mg/kg) RT high-dosedeuterium oxide + cisplatin group 4.08 ± 1.22* (Ringer's deuterium oxidesolution40 ml/kg + cisplatin 0.6 mg/kg) Hyperthermic low-dose deuteriumoxide + cisplatin  3.11 ± 0.56** group (Ringer's deuterium oxidesolution 5 ml/kg + cisplatin0.6 mg/kg); Hyperthermic mid-dose deuteriumoxide + cisplatin  2.49 ± 0.62** group (Ringer's deuterium oxidesolution20 ml/kg + cisplatin0.6 mg/kg); Hyperthermic high-dose deuteriumoxide + cisplatin  0.44 ± 0.46** group (Ringer's deuterium oxidesolution40 ml/kg + cisplatin0.6 mg/kg). Compared to the negative controlgroup: *P < 0.001, **P < 0.0001. RT means room temperature.

Cisplatin is a representative anti-tumor platinum preparation, anddeuterium oxide has a broad-spectrum effect of enhancing anti-tumorefficacy. The results demonstrate that the 42° C. hyperthermicintraperitoneal lavage with deuterium oxide in combination withcisplatin could significantly extend the survival time, inhibitproduction of ascitic fluid than deuterium oxide under room temperaturein mice bearing H22 ascites tumors. The application of hyperthermicdeuterium oxide exhibits an unexpected anti-tumor effect of enhancing,thereby improves quality of life of the tumor-bearing mice.

Example 20

Inhibition of sarcoma S180 (ascites type) in tumor-bearing mice by 42±1°C. hyperthermic intraperitoneal retention lavage using deuterium oxidein combination with oxaliplatin and using deuterium oxide in combinationwith oxaliplatin and mitomycin.

Experimental Method:

Animal: 4 to 5 week-aged male Kunming mice, provided by ShanghaiSIPPR-BK Laboratory Animal Co. Ltd.

Cell lines: sarcoma S180 (ascites type) cell line was provided byNANJING KEYGEN BIOTECH. CO., LTD.

Modeling: 0.2 ml suspension of sarcoma S180 cells containing 1×10⁷cells/ml was inoculated into the peritoneal cavity of mice.

Test drugs: oxaliplatin, mitomycin, and the glucose deuterium oxidesolution as prepared in Example 2.

Grouping and dosing: the mouse model was established 7 to 8 days afterinoculation, and the mice were randomized with 12 animals/group.

Oxaliplatin and mitomycin were dissolved in a 42±1° C. glucose deuteriumoxide solution, and used to lavage the peritoneal cavity of thetumor-bearing mice.

1). Blank control group (5% glucose injection, 5 ml/kg);2). Chemo group (oxaliplatin 0.8 mg/kg+5% glucose injection, 5 ml/kg);3). Low-dose deuterium oxide+oxaliplatin group (5% glucose deuteriumoxide solution, 5 ml/kg+oxaliplatin 0.8 mg/kg);4). Mid-dose deuterium oxide+oxaliplatin group (5% glucose deuteriumoxide solution, 20 ml/kg+oxaliplatin 0.8 mg/kg);5). High-dose deuterium oxide+oxaliplatin group (5% glucose deuteriumoxide solution, 40 ml/kg+oxaliplatin 0.8 mg/kg);6). Low-dose deuterium oxide+oxaliplatin+mitomycin group (42±1° C. 5%glucose deuterium oxide solution, 5 ml/kg+oxaliplatin 0.8mg/kg+mitomycin 0.4 mg/kg);7). Mid-dose deuterium oxide+oxaliplatin+mitomycin group (42±1° C. 5%glucose deuterium oxide solution, 20 ml/kg+oxaliplatin 0.8mg/kg+mitomycin 0.4 mg/kg);8). High-dose deuterium oxide+oxaliplatin+mitomycin group (42±1° C. 5%glucose deuterium oxide solution, 40 ml/kg+oxaliplatin 0.8mg/kg+mitomycin 0.4 mg/kg).

The peritoneal cavity was lavaged with the 42±1° C. glucose deuteriumoxide solution in combination with the above composition, which wereretained for 10 min in the peritoneal cavity after being introduced,which was repeated for 3 times. The lavage was performed once anotherday for 5 successive times. The body weight and abdomen circumference ofmice were measured on each day before administration of the drugs, andthe daily living status of mice was observed. 24 hours after theadministration of drugs was completed (on day 11), 4 mice from eachgroup were sacrificed, the volume of ascitic fluid was measured, andbodies of the mice were dissected to observe the peritoneal organs andmetastasis to lung. The rest of mice were observed for their survivaltime, and the extension of life was calculated.

4. Observation of indicators: the same as Example 18.5. Statistics: the same as Example 18.

6. Results

TABLE 16 Effect of 42 ± 1° C. intraperitoneal lavage with deuteriumoxide in combination with oxaliplatin and mitomycin on survival time (indays) of animals(x ± SD, n = 8) Group MST(days) T/C(%) Blank controlgroup (glucose injection, 5 ml/kg) 12.2 ± 3.0  Chemo group (oxaliplatin0.8 mg/kg + glucose 21.7 ± 2.8* 177.6 injection 5 ml/kg) Low-dosedeuterium oxide + oxaliplatin(glucose 23.4 ± 3.2* 191.8 deuterium oxidesolution5 ml/kg + oxaliplatin 0.8 mg/kg) Mid-dose deuterium oxide +oxaliplatin(glucose 27.7 ± 4.9* 227.0 deuterium oxide solution 20ml/kg + oxaliplatin 0.8 mg/kg) High-dose deuterium oxide +oxaliplatin(glucose 31.1 ± 6.1* 254.9 deuterium oxide solution 40ml/kg + oxaliplatin 0.8 mg/kg) Low-dose deuterium oxide + oxaliplatin +26.4 216.3 mitomycin(glucose deuterium oxide solution 5 ml/kg +oxaliplatin 0.8 mg/kg + mitomycin 0.4 mg/kg) Mid-dose deuterium oxide +oxaliplatin + 30.1 246.7 mitomycin(glucose deuterium oxide solution 20ml/kg + oxaliplatin 0.8 mg/kg + mitomycin 0.4 mg/kg) High-dose deuteriumoxide + oxaliplatin + 32.8 268.8 mitomycin(glucose deuterium oxidesolution 40 ml/kg + oxaliplatin 0.8 mg/kg + mitomycin 0.4 mg/kg)Compared to the negative control group: *P < 0.0001.

TABLE 17 Effect of 42 ± 1° C. intraperitoneal lavage with deuteriumoxide in combination with oxaliplatin and mitomycin on the volume ofascitic fluid(x ± SD, n = 8) Ascitic Group fluid (ml) Blank controlgroup (glucose injection, 5 ml/kg) 16.5 ± 4.3  Chemo group(oxaliplatin0.8 mg/kg + glucose 8.7 ± 1.9* injection 5 ml/kg) Low-dosedeuterium oxide + oxaliplatin(oxaliplatin 8.1 ± 2.8* 0.8 mg/kg + glucosedeuterium oxide solution 5 ml/kg) Mid-dose deuterium oxide +oxaliplatin(oxaliplatin 7.3 ± 2.9* 0.8 mg/kg + glucose deuterium oxidesolution 20 ml/kg) High-dose deuterium oxide + oxaliplatin(oxaliplatin 2.1 ± 1.3** 0.8 mg/kg + glucose deuterium oxide solution 40 ml/kg)Low-dose deuterium oxide + oxaliplatin + 7.5 ± 3.1*mitomycin(oxaliplatin 0.8 mg/kg + mitomycin 0.4 mg/kg + glucosedeuteriumoxide solution 5 ml/kg) Mid-dose deuterium oxide + oxaliplatin +  3.7 ±1.6** mitomycin(oxaliplatin 0.8 mg/kg + mitomycin 0.4 mg/kg + glucosedeuterium oxide solution 20 ml/kg) High-dose deuterium oxide +oxaliplatin +  1.1 ± 0.5** mitomycin(oxaliplatin 0.8 mg/kg + mitomycin0.4 mg/kg + glucose deuterium oxide solution 40 ml/kg) Compared to thenegative control group: *P < 0.0001, **P < 0.00001.

Oxaliplatin is a third-generation anti-tumor platinum preparation,mitomycin is an anti-tumor antibiotic to which tumor cells in G0 to Sphases are sensitive, and deuterium oxide has a broad-spectrum effect ofenhancing anti-tumor efficacy, to which tumor cells in the S phase arealso sensitive. Their combination can further enhance anti-tumorefficacy. The results demonstrate that the hyperthermic deuterium oxidein combination with oxaliplatin and mitomycin can also significantlyextend the survival time of mice bearing sarcoma S180, and inhibitsproduction of ascitic fluid in mice bearing sarcoma S180. This indicatesthat combination of hyperthermic deuterium oxide with platinumpreparation and anti-tumor antibiotics has general applicability ininhibition of growth of various tumors.

Example 21

Inhibition of growth of xenograft tumors from human colon cancer HCT-166cells in nude mice by intravenous injection of deuterium oxide incombination with 5-fluorouracil

Animal: 6 to 7 week-aged female BALB/c nude mice, each weighing 18 to 20g, provided by Shanghai SLAC Laboratory Animal Co. Ltd.

Cell lines: human colon cancer cells HCT-116 were provided by NANJINGKEYGEN BIOTECH. CO., LTD.

Test drugs: 5-fluorouracil (5-FU), and the glucose (5%) deuterium oxidesolution (containing 5 g glucose per 100 ml) prepared in Example 2.

Experimental Method: 1. Modeling

Tumor cells were resuscitated and cultured according to a routineprocedure, a suspension of cultured HCT-116 cells was collected, sterilesodium chloride physiological solution was added thereto to make asuspension at a concentration of 1×10⁷ cells/ml, and 0.1 ml of thesuspension was subcutaneously inoculated into the right axillary fossaof each nude mouse.

2. Grouping and Dosing Scheme

The diameter of xenograft tumors in the nude mice was measured with avernier caliper, and 10 days after inoculation, when tumors grew to 100to 110 cm³, the animals were randomized with 8 mice per group. Meanwhiledrugs were administered by injection into tail vein, once every threedays, for 21 successive days. By measuring the tumor diameter, theanti-tumor efficacy of test samples was dynamically observed, and thebody weight of animals was also recorded to observe the toxicity of thetest samples. 21 days after administration, the nude mice weresacrificed, and tumors were harvested by surgery, weighed andphotographed. Bone marrow nucleated cells were isolated by a routinemethod, DNA was extracted from the cells, the OD value was measured in aUV-260 spectrometer as a representative of the DNA content, andinhibition of DNA synthesis by the test samples was observed.

The test used 7 groups in total, with 8 animals per group.

1) Negative control group: 5% glucose injection, 10 ml/kg;2) Paclitaxel positive control group: paclitaxel (8 mg/kg) diluted insodium chloride physiological solution;3) High-dose 5-FU positive control group: 5-FU (30 mg/kg) diluted in 5%glucose injection;4) Low-dose 5-FU positive control group: 5-FU (12 mg/kg) diluted in 5%glucose injection.5) Test group 1: 5-FU (12 mg/kg) diluted in the glucose deuterium oxidesolution (5 ml/kg).6) Test group 2: 5-FU (12 mg/kg) diluted in the glucose deuterium oxidesolution (10 ml/kg).7) Test group 3: 5-FU (12 mg/kg) diluted in the glucose deuterium oxidesolution (20 ml/kg).

3. Observation of Indicators 1) Anti-Tumor Activity Evaluation byRelative Tumor Volume

Tumor volume (TV) was calculated by the following equation:

TV=½×a×b ²,

wherein a and b refer to length and width, respectively.

From the measurements, relative tumor volume (RTV) was calculated by thefollowing equation:

RTV=V _(t) /V ₀

wherein V₀ is the tumor volume measured on initial administration (i.e.do), and V_(t) is the tumor volume measured each time afterwards.

Relative tumor growth ratio T/C (%) was calculated by the followingequation:

${{TC}(\%)} = {\frac{T_{RTV}}{C_{RTV}} \times 100}$

T_(RTV): RTV of the treatment group; C_(RTV): RTV of the negativecontrol group.

Criteria of efficacy evaluation: T/C %>40%, not efficacious; T/C %≦40%and statistically P<0.05, efficacious.

2) Anti-Tumor Activity Evaluation by Tumor Weight

Inhibition of tumor growth (%) was calculated by the following equation:

${{Inhibition}\mspace{14mu} {of}\mspace{14mu} {tumor}\mspace{14mu} {growth}} = {\frac{{ATW}_{NCG} - {ATW}_{DG}}{{ATW}_{NCG}} \times 100\%}$

ATW_(NCG): Average tumor weight in negative control group

ATW_(DG): Average tumor weight in drug-dosed group

Criteria of efficacy evaluation: Inhibition of tumor growth<40%, notefficacious; Inhibition of tumor growth≧40% and statistically P<0.05,efficacious.

3) Observation of Drug's Inhibition of Bone Marrow and Monitoring ofDrug's Toxicity Based on Bone Marrow DNA Content. 4. Statistics

The mean value was expressed in X±SD. Inter-group analysis wasstatistically performed with t test. The results were statisticallyanalyzed using SPSS (Statistical Package for the Social Science) 17.0.

5. Results

Anti-tumor activity evaluation by xenograft tumor volume: the resultsdemonstrate that deuterium oxide in combination with low-dose 5-FUshowed a good inhibitory effect on the xenograft tumors; 21 days afteradministration, the tumor volumes were 1.22±0.15 cm³ in the groupreceiving the deuterium oxide solution (5 ml/kg) in combination with5-FU, 1.45±0.25 cm³ in the group receiving the deuterium oxide solution(10 ml/kg) in combination with 5-FU, and 1.27±0.24 cm³ in the groupreceiving the deuterium oxide solution (20 ml/kg) in combination with5-FU, all with a T/C<40% as compared to the negative control group, andsatisfied the criteria for pharmacological efficacy. But the groupreceiving low-dose 5-FU alone showed a reduced tumor volume to 2.27±0.30cm³ only, with a T/C of 80.91%, which is >40% and did not satisfy thecriteria for pharmacological efficacy. Therefore, when combined with5-FU, deuterium oxide significantly enhance the anti-tumor efficacy of5-FU, as shown in FIG. 5.

Anti-tumor activity evaluation by xenograft tumor weight: the resultsdemonstrate that deuterium oxide in combination with low-dose 5-FUshowed a good inhibitory effect on the xenograft tumors; 21 days afteradministration, the inhibitions were 56.32% in the group receiving thedeuterium oxide solution (5 ml/kg) in combination with 5-FU, 53.70% inthe group receiving the deuterium oxide solution (10 ml/kg) incombination with 5-FU, and 54.22% in the group receiving the deuteriumoxide solution (20 ml/kg) in combination with 5-FU, all with a T/C>40%and P<0.001 as compared to the tumor weight in the negative controlgroup, and all satisfied the criteria for pharmacological efficacy. Thegroup receiving low-dose 5-FU alone showed an inhibition of 23.64% anddid not satisfy the criteria for pharmacological efficacy. Therefore,when combined with 5-FU, deuterium oxide significantly enhance theanti-tumor efficacy of 5-FU, as shown in Table 18 and FIG. 6.

TABLE 18 Effect of deuterium oxide in combination with 5-FU on xenografttumors from human colon cancer HCT-116 cells in nude mice(x ± SD, n = 8)Bone marrow Body weight (g) Tumor weight Inhibition DNA content GroupDose × Times (Initial/End) (g) (%) (OD) Negative Control 10 ml/kg18.5/+0.4 2.92 ± 0.27 — 0.950 ± 0.2711 Paclitaxel positive 8 mg/kg × 1118.4/−1.2 0.94 ± 0.25 67.80%*  0.841 ± 0.3014** control 5-FU positivecontrol 30 mg/kg × 11 18.4/−1.0 1.04 ± 0.13 64.38%*  0.836 ± 0.5023**5-FU(H) 5-FU low-dose 12 mg/kg × 11 18.0/−0.6 2.23 ± 0.2  23.63%  0.897± 0.3783** 5-FU(L) Test group 1 Glucose deuterium 18.3/−0.4 1.34 ± 0.3854.10%* 0.913 ± 0.5122 5-FU(L) + HW(L) oxide solution (5 ml/kg) +5-FU(12 mg/kg) × 11 Test group 2 Glucose deuterium 18.5/+0.2 1.35 ± 0.3453.76%* 0.901 ± 0.4655 5-FU(L) + HW(M) oxide solution(10 ml/kg) +5-FU(12 mg/kg) × 11 Test group 3 Glucose deuterium 18.4/+0.2 1.28 ± 0.4656.16%* 0.921 ± 0.2910 5-FU(L) + HW(H) oxide solution(20 ml/kg) +5-FU(12 mg/kg) × 11 Compared to the negative control group: *P < 0.001,**P < 0.05. Inhibition of tumor growth (%) >40% indicatespharmacological efficacy.

Toxicity reduction: importantly, in the high-dose paclitaxel and 5-FUpositive control groups, despite apparent inhibition of tumor growth,the body weight of tumor-bearing mice dropped greatly from 18.4±0.5 g to16.8±0.9 g and to 17.4±0.5 g, respectively, and the bone marrow DNAcontent also decreased, indicating significant toxicity of paclitaxeland 5-FU to animals. The deuterium oxide solution in combination with5-FU (10 mg/kg, one third of that of control) can achieve the sameefficacy as high-dose paclitaxel and 5-FU (30 mg/kg) (equivalence),while the body weight of the tumor-bearing mice during dosing did notdrop, and the bone marrow DNA content did not decrease, without thetoxicity caused by high-dose paclitaxel and 5-FU. Because toxicity ofanti-tumor drugs is very harmful to patients and even unacceptable tosome weak or aged patients, chemotherapeutic drugs having low toxic sideeffects and a strong killing effect are urgently needed in tumortreatment. Hence, deuterium oxide in combination with low-dose 5-FU hasa great advantage in clinical application, as shown in Table 18 and FIG.7.

Example 22

Inhibition of growth of xenograft tumors from human breast cancer MCF-7cells in nude mice by intravenous injection of deuterium oxide incombination with gemcitabine

Animal: 6 to 7 week-aged female BALB/c nude mice, each weighing 18 to 20g, provided by Shanghai SLAC Laboratory Animal Co. Ltd.

Cell lines: human breast cancer MCF-7 cells were provided by NANJINGKEYGEN BIOTECH. CO., LTD.

Test drugs: gemcitabine (GEM) and the sodium chloride deuterium oxidesolution prepared in Example 1.

Experimental Method: 1. Modeling

Tumor cells were resuscitated and cultured according to a routineprocedure, suspension of cultured MCF-7 cells was collected, sterilesodium chloride physiological solution was added thereto to make asuspension at a concentration of 1×10⁷ cells/ml, and 0.1 ml of thesuspension was subcutaneously inoculated into the right axillary fossaof each nude mouse.

2. Grouping and Dosing Scheme

The diameter of xenograft tumors in the nude mice was measured with avernier caliper, and 10 days after inoculation, when tumors grew to 100to 110 cm³, the animals were randomized with 8 mice per group. Meanwhiledrugs were administered by injection into tail vein, once every threedays, for 21 successive days. By measuring the tumor diameter, theanti-tumor efficacy of test samples was dynamically observed. Meanwhilethe body weight of animals was also recorded to observe the toxicity ofthe test samples. 21 days after administration, the nude mice weresacrificed, and tumors were harvested by surgery, weighed andphotographed. Bone marrow nucleated cells were isolated by a routinemethod, DNA was extracted from the cells, the OD value was measured in aUV-260 spectrometer as a representative of the DNA content, andinhibition of DNA synthesis by the test samples was observed.

The test used 7 groups in total, with 8 animals per group.

The test used 7 groups:

1) Negative control group: sodium chloride physiological solution, 10ml/kg;2) Paclitaxel positive control group: paclitaxel diluted in sodiumchloride physiological solution (8 mg/kg);3) Gemcitabine positive control group: gemcitabine diluted in sodiumchloride physiological solution (20 mg/kg);4) Low-dose gemcitabine control group: gemcitabine diluted in sodiumchloride physiological solution (10 mg/kg);5) Test group 1: gemcitabine (10 mg/kg) diluted in the sodium chloridedeuterium oxide solution (5 ml/kg).6) Test group 2: gemcitabine (10 mg/kg) diluted in the sodium chloridedeuterium oxide solution (10 ml/kg).7) Test group 3: gemcitabine (10 ml/kg) diluted in the sodium chloridedeuterium oxide solution (20 ml/kg).3. Observation of indicators: the same as Example 21.4. Statistics: the same as Example 21.

5. Results

Anti-tumor activity evaluation by xenograft tumor volume: the sodiumchloride deuterium oxide solution in combination with gemcitabine 10mg/kg showed a good inhibitory effect on the xenograft tumors; 21 daysafter administration, the tumor volumes were 1.949±0.491 cm³ in thegroup receiving the deuterium oxide solution 5 ml/kg in combination withgemcitabine, 1.743±0.503 cm³ in the group receiving the deuterium oxidesolution 10 ml/kg in combination with gemcitabine, and 1.671±0.386 cm³in the group receiving the deuterium oxide solution 20 ml/kg incombination with gemcitabine, all with a T/C<40% as compared to thenegative control group, and satisfied the criteria for pharmacologicalefficacy. The group receiving gemcitabine 10 mg/kg alone showed only aslightly reduced tumor volume of 2.757±0.342 cm³ with a T/C of 60.39%,which was >40% and did not satisfy the criteria for pharmacologicalefficacy. When combined with gemcitabine, deuterium oxide significantlyenhances the anti-tumor efficacy of gemcitabine, as shown in FIG. 8.

Anti-tumor activity evaluation by xenograft tumor weight: the deuteriumoxide solution in combination with gemcitabine showed a good inhibitoryeffect on the xenograft tumors; 21 days after administration, theinhibitions were 48.82% in the group receiving the deuterium oxidesolution 5 ml/kg in combination with gemcitabine, 57.78% in the groupreceiving the deuterium oxide solution 10 ml/kg in combination withgemcitabine, and 60.70% in the group receiving the deuterium oxidesolution 20 ml/kg in combination with gemcitabine, all with a T/C>40%and P<0.001 as compared to the negative control group, and all satisfiedthe criteria for pharmacological efficacy. The group receivinggemcitabine 10 mg/kg alone showed only a slightly reduced tumor weightand an inhibition of 39.18%, which was less than 40% and did not satisfythe criteria for pharmacological efficacy. Therefore, deuterium oxidesignificantly enhances the anti-tumor efficacy of gemcitabine, as shownin Table 19 and FIG. 9.

TABLE 19 Effect of deuterium oxide in combination with gemcitabine onxenograft tumors from human breast cancer MCF-7 cells in nude mice (x ±SD, n = 8) Body Bone marrow weight Tumor weight Inhibition DNA contentGroup Dose × Times (g) (Initial/End) (g) (%) (OD) Negative Control 10ml/kg × 11 18.3/+3.3 4.07 ± 0.76 — 0.931 ± 0.2011 Paclitaxel positive 8mg/kg × 11 18.3/+1.0 1.59 ± 0.40 60.93%* 0.881 ± 0.2502 controlGemcitabine positive 20 mg/kg × 11 18.1/+0.9 1.57 ± 0.21 61.42%* 0.823 ±0.1416** control GEM(H) Gemcitabine low-dose 10 mg/kg × 11 18.0/+2.92.48 ± 0.41 39.06% 0.908 ± 0.2543 GEM(L) Test group 1 Sodium chloride18.4/+3.1 2.08 ± 0.28 48.89%* 0.923 ± 0.3101 GEM(L) + HW(L) deuteriumoxide solution (5 ml/kg) + GEM (10 mg/kg) × 11 Test group 2 Sodiumchloride 18.0/+3.3 1.72 ± 0.51 57.73%* 0.942 ± 0.2896 GEM(L) + HW(M)deuterium oxide solution (10 ml/kg) + GEM (10 mg/kg) × 11 Test group 3Sodium chloride 18.4/+2.4  1.6 ± 0.55 60.68%* 0.920 ± 0.4167 GEM(L) +HW(H) deuterium oxide solution (20 ml/kg) + GEM (10 mg/kg) × 11 Comparedto the negative control group: *P < 0.001, **P < 0.05. Inhibition oftumor growth (%) >40% indicates pharmacological efficacy.

Toxicity reduction: importantly, during the experiment, the average bodyweight of tumor-bearing mice in the negative control group increased by3.3 g; in the paclitaxel and gemcitabine positive control groups,despite apparent reduction in tumor weight, the body weight oftumor-bearing mice only slightly increased, and the bone marrow DNAcontent also only slightly increased, indicating certain toxicity ofpaclitaxel and gemcitabine to animals. The sodium chloride deuteriumoxide solution in combination with low-dose gemcitabine (10 mg/kg, halfof that of control) can achieve the same efficacy as high-dosepaclitaxel (8 mg/kg) and high-dose gemcitabine (20 mg/kg) (equivalence),while the body weight of the tumor-bearing mice increased similarly tothe negative control group, and the bone marrow DNA content alsoincreased, without the toxicity caused by high-dose paclitaxel andgemcitabine. Toxicity of anti-tumor drugs is very harmful to patientsand even unacceptable to some weak or aged patients, and thus causes aserious problem to clinical anti-tumor chemotherapy. Hence, deuteriumoxide in combination with low-dose gemcitabine has a great advantage inclinical applications, as shown in Table 19 and FIG. 10.

5-FU and gemcitabine are antimetabolites among cytotoxic anti-tumordrugs. It can be also seen in Examples 19 and 20 that deuterium oxide incombination with antimetabolite anti-tumor drugs has generalapplicability for inhibition of growth of various tumors.

Example 23

Inhibition of growth of xenograft tumors on nude mice-bearing human lungcancer A549 cell by intravenous injection of deuterium oxide incombination with gemcitabine

Animal: 6 to 7 week-aged female BALB/c nude mice, each weighing 18 to 20g, provided by Shanghai SLAC Laboratory Animal Co. Ltd.

Cell lines: human lung cancer A549 cells were provided by NANJING KEYGENBIOTECH. CO., LTD.

Test drugs: gemcitabine (GEM) and the sodium chloride deuterium oxidesolution prepared in Example 1.

1. Tumor cells were resuscitated and inoculated, and sterile sodiumchloride physiological solution was added thereto to make a cellsuspension which was subcutaneously inoculated into the upper rightaxillary fossa of animals. 5×10⁶ tumor cells were inoculated into eachanimal.

2. Grouping and Dosing Scheme

The diameter of xenograft tumors in the nude mice was measured with avernier caliper, and 10 days after inoculation, when tumors grew to 100to 110 cm³, the animals were randomized with 8 mice per group. Meanwhiledrugs were administered by injection into tail vein, once every threedays, for 21 successive days. By measuring the tumor diameter, theanti-tumor efficacy of test samples was dynamically observed. Meanwhilethe body weight of animals was also recorded to observe the toxicity ofthe test samples. 21 days after administration, the nude mice weresacrificed, and tumors were harvested by surgery, weighed andphotographed.

The test used 7 groups:

1) Negative control group: sodium chloride physiological solution, 10ml/kg;2) Paclitaxel positive control group: paclitaxel diluted in sodiumchloride physiological solution (8 mg/kg);3) Gemcitabine positive control group: gemcitabine diluted in sodiumchloride physiological solution (20 mg/kg);4) Low-dose gemcitabine control group: gemcitabine diluted in sodiumchloride physiological solution (10 mg/kg);5) Test group 1: gemcitabine (10 mg/kg) diluted in the sodium chloridedeuterium oxide solution (5 ml/kg).6) Test group 2: gemcitabine (10 mg/kg) diluted in the sodium chloridedeuterium oxide solution (10 ml/kg).7) Test group 3: gemcitabine (10 ml/kg) diluted in the sodium chloridedeuterium oxide solution (20 ml/kg).3. Observation of indicators: the same as Example 21.4. Statistics: the same as Example 21.

5. Results

Anti-tumor activity evaluations by xenograft tumor volume and weight:the results demonstrate that, 21 days after administration, as comparedto the sodium chloride physiological solution control group, the groupreceiving low-dose gemcitabine alone showed a reduced tumor weight to2.23±0.2 g and an inhibition of 23.6%, which however did not satisfy thecriteria for pharmacological efficacy. As compared to the control group,the deuterium oxide solution in combination with gemcitabine all showeda good inhibitory effect on the xenograft tumors; the group receivingthe deuterium oxide solution 5 ml/kg in combination with gemcitabineshowed a tumor weight of 1.34±0.38 g and a tumor proliferation ratio of54.22%, the group receiving the deuterium oxide solution 10 ml/kg incombination with gemcitabine showed a tumor weight of 1.35±0.34 g and atumor proliferation ratio of 53.70%, and the group receiving thedeuterium oxide solution 20 ml/kg in combination with gemcitabine showeda tumor weight of 1.28±0.46 g and a tumor proliferation ratio of 56.32%,all having P<0.001 and satisfying the criteria for pharmacologicalefficacy, as shown in FIGS. 11 and 12.

TABLE 20 Effect of sodium chloride deuterium oxide solution on tumorweight of human lung cancer xenograft tumors in nude mice (x ± SD, n =8) Body weight Tumor weight Inhibition Group Dose × Times (g)(Initial/End) (g) (%) Negative control 10 ml/kg 18.5/+1.4 2.92 ± 0.27 —Paclitaxel positive 8 mg/kg × 11 18.4/+0.9 0.94 ± 0.25 67.97* controlGemcitabine positive 20 mg/kg × 11 18.4/+0.5 1.04 ± 0.13 64.54* controlGEM(H) Gemcitabine low-dose 10 mg/kg × 11 18.0/+0.4 2.23 ± 0.2  23.64GEM(L) Test group 1 Sodium chloride 18.3/+1.5 1.34 ± 0.38 54.22*GEM(L) + HW(L) deuterium oxide solution (5 ml/kg) + GEM (10 mg/kg) × 11Test group 2 Sodium chloride 18.5/+1.1 1.35 ± 0.34 53.70* GEM(L) + HW(M)deuterium oxide solution (10 ml/kg) + GEM (10 mg/kg) × 11 Test group 3Sodium chloride 18.4/+0.7 1.28 ± 0.46 56.32* GEM(L) + HW(H) deuteriumoxide solution (20 ml/kg) + GEM (10 mg/kg) × 11 As compared to thecontrol group, *P < 0.001, and inhibition of tumor weight >40% indicatespharmacological efficacy.

Importantly, in the paclitaxel and gemcitabine positive control groups,despite an apparent reduction in tumor weight to 0.94±0.25 g and1.04±0.13 g, respectively, and a tumor proliferation ratio of 67.97% and64.54%, respectively, the body weight of tumor-bearing mice barelyincreased, indicating significant toxicity of paclitaxel and gemcitabineto animals. Deuterium oxide in combination with low-dose gemcitabine canachieve the same efficacy as high-dose paclitaxel and gemcitabine, doesnot cause the corresponding toxicity, and results in an increased bodyweight of tumor-bearing mice during administration, as shown in Table 20and FIG. 13.

Example 24

Inhibition of growth of xenograft tumors from human ovarian cancer SKOV3cells in nude mice by intravenous injection of deuterium oxide incombination with paclitaxel

Animal: 6 to 7 week-aged female BALB/c nude mice, each weighing 18 to 20g, provided by Shanghai SLAC Laboratory Animal Co. Ltd.

Cell lines: human ovarian cancer SKOV3 cells were provided by NANJINGKEYGEN BIOTECH. CO., LTD.

Test drugs: paclitaxel and the sodium chloride deuterium oxide solution,prepared in Example 1.

Experimental Method: 1. Tumor Cell Resuscitation and Inoculation

Tumor cells were resuscitated and inoculated, and sterile sodiumchloride physiological solution was added thereto to make a cellsuspension which was subcutaneously inoculated into the upper rightaxillary fossa of animals. 5×10⁶ tumor cells were inoculated into eachanimal.

2. Grouping and Dosing Scheme

The diameter of xenograft tumors in the nude mice was measured with avernier caliper, and 10 days after inoculation, when tumors grew to 100to 110 cm³, the animals were randomized. Meanwhile drugs wereadministered by injection into tail vein, once every three days, for 21successive days. The body weight of animals was also recorded to observethe toxicity of the test samples. 21 days after administration, the nudemice were sacrificed, and tumors were harvested by surgery, and weighed.

The test used 6 groups in total, with 5 animals per group.

1) Control group: sodium chloride physiological solution, 10 ml/kg;2) Paclitaxel positive control group: paclitaxel (8 mg/kg) diluted insodium chloride physiological solution;3) Sodium chloride deuterium oxide solution (10 ml/kg);4) Test group 1: paclitaxel (8 mg/kg) diluted in the sodium chloridedeuterium oxide solution (5 ml/kg);5) Test group 2: paclitaxel (8 mg/kg) diluted in the sodium chloridedeuterium oxide solution (10 ml/kg);6) Test group 3: paclitaxel (8 mg/kg) diluted in the sodium chloridedeuterium oxide solution (20 ml/kg).3. Observation of indicators: the same as Example 21.4. Statistics: the same as Example 21.

The results are shown in Table 21.

TABLE 21 Effect of sodium chloride deuterium oxide solution incombination with paclitaxel on tumor weight of xenograft tumors fromhuman ovarian cancer SKOV3 cells in nude mice (x ± SD, n = 5) Bodyweight Tumor weight Inhibition Group Dose × Times (g) (Initial/End) (g)(%) Control 10 ml/kg 20.8/+4.0 3.22 ± 1.59 — Paclitaxel 8 mg/kg × 1120.1/+2.8 2.07 ± 1.06 35.7 Sodium chloride 10 ml/kg × 11 21.1/+3.9 2.44± 1.51 24.2 deuterium oxide solution Test group 1 Sodium chloride21.0/+3.8 1.95 ± 0.57 39.4 Sodium chloride deuterium oxide deuteriumoxide solution (5 ml/kg) + solution + Paclitaxel Paclitaxel (8 mg/kg) ×11 Test group 2 Sodium chloride 20.4/+3.7 1.71 ± 0.87 46.8* Sodiumchloride deuterium oxide deuterium oxide solution (10 ml/kg) +solution + Paclitaxel Paclitaxel (8 mg/kg) × 11 Test group 3 Sodiumchloride 20.9/+4.1 1.45 ± 0.42 54.9* Sodium chloride deuterium oxidedeuterium oxide solution (20 ml/kg) + solution + Paclitaxel Paclitaxel(8 mg/kg) × 11 Compared to the control group: *P < 0.01.

Paclitaxel is an important phytogenic anti-tumor drug, having varioustypes of a similar mechanism of action, including paclitaxel, paclitaxelliposomes, paclitaxel albumin, and docetaxel. The results demonstratethat, 21 days after administration, as compared to the control group,the groups receiving the deuterium oxide solution (10 ml/kg and 20ml/kg) in combination with paclitaxel (8 mg/kg) showed a good inhibitoryeffect on xenograft tumors, with a significant reduction in tumorweight, while the tumor inhibition T/C was 54.9% on day 21 with P<0.001.On the other hand, as compared to the control group, the group receivingpaclitaxel (8 mg/kg) alone showed a reduction, but did not satisfy thecriteria of >40% for pharmacological efficacy. As compared to thecontrol group, the group receiving the deuterium oxide solution (5ml/kg) in combination with paclitaxel (12 mg/kg) showed a reduction, butdid not satisfy the criteria of >40% for pharmacological efficacy.Deuterium oxide as a broad-spectrum anti-tumor efficacy-enhancing agentcan also enhance the efficacy of phytogenic anti-tumor drugs.

Example 25

Inhibition of growth of xenograft tumors from human bladder cancer 5637cells in nude mice by intravenous injection of deuterium oxide incombination with thiotepa

Animal: 6 to 7 week-aged female BALB/c nude mice, each weighing 18 to 20g, provided by Shanghai SLAC Laboratory Animal Co. Ltd.

Cell lines: human bladder cancer 5637 cells were provided by NANJINGKEYGEN BIOTECH. CO., LTD.

Test drugs: thiotepa and the sodium chloride deuterium oxide solution,prepared in Example 1.

Experimental Method: 1. Tumor Cell Resuscitation and Inoculation

Tumor cells were resuscitated and inoculated, and sterile sodiumchloride physiological solution was added thereto to make a cellsuspension which was subcutaneously inoculated into the upper rightaxillary fossa of animals. 5×10⁶ tumor cells were inoculated into eachanimal.

2. Grouping and Dosing Scheme

The diameter of xenograft tumors in the nude mice was measured with avernier caliper, and 10 days after inoculation, when tumors grew to 100to 110 cm³, the animals were randomized. Meanwhile drugs wereadministered by injection into tail vein, once every three days, for 21successive days. The body weight of animals was also recorded to observethe toxicity of the test samples. 21 days after administration, the nudemice were sacrificed, and tumors were harvested by surgery, and weighed.

The test used 5 groups in total, with 5 animals per group.

1) Control group: sodium chloride physiological solution, 10 ml/kg;2) Thiotepa positive control group: thiotepa (0.2 mg/kg) diluted insodium chloride physiological solution;3) Test group 1: thiotepa (0.2 mg/kg) diluted in the sodium chloridedeuterium oxide solution (1 ml/kg);4) Test group 2: thiotepa (0.2 mg/kg) diluted in the sodium chloridedeuterium oxide solution (10 ml/kg);5) Test group 3: thiotepa (0.2 mg/kg) diluted in the sodium chloridedeuterium oxide solution (25 ml/kg).3. Observation of indicators: the same as Example 21.4. Statistics: the same as Example 21.

The results demonstrate that, 21 days after administration, as comparedto the control group, the groups receiving the two dosages of deuteriumoxide solution (10 ml/kg and 20 ml/kg) in combination with thiotepa (0.2mg/kg) showed a good inhibitory effect on xenograft tumors, with asignificant reduction in tumor weight, while the tumor inhibitions T/Cwere 47.0% and 63.4% on day 21, with P<0.01 and P<0.001, respectively.On the other hand, as compared to the control group, the group receivingthiotepa (0.2 mg/kg) alone did not show a considerable difference. Ascompared to the control group, the group receiving the deuterium oxidesolution (1 ml/kg) in combination with thiotepa (0.2 mg/kg) did not showa considerable difference. The results are shown in Table 22.

TABLE 22 Effect of sodium chloride deuterium oxide solution incombination with thiotepa on tumor weight of xenograft tumors from humanbladder cancer 56373 cells in nude mice (x ± SD, n = 5) Body weightTumor weight Inhibition Groups Dose × Times (g) (Initial/End) (g) (%)Control 10 ml/kg 19.8/+3.1 2.87 ± 1.11 — Thiotepa 0.2 mg/kg × 1120.6/+2.6 1.75 ± 1.06 39.0 Test group 1 Sodium chloride 20.4/+3.3 1.72 ±0.73 40.1 Sodium chloride deuterium deuterium oxide oxide solution +thiotepa solution (1 ml/kg) + thiotepa (0.2 mg/kg) × 11 Test group 2Sodium chloride 20.1/+3.0 1.32 ± 0.55 54.0* Sodium chloride deuteriumdeuterium oxide oxide solution + thiotepa solution (10 ml/kg) + thiotepa(0.2 mg/kg) × 11 Test group 3 Sodium chloride 19.9/+4.1 1.05 ± 0.3863.4** Sodium chloride deuterium deuterium oxide oxide solution +thiotepa solution (25 ml/kg) + thiotepa (0.2 mg/kg) × 11 Compared to thecontrol group: *P < 0.01, **P < 0.001.

Thiotepa is an ethyleneimine alkylating agent involved in guaninebinding and affecting DNA synthesis, and is effective on various typesof tumor cells, especially bladder cancer cells. Deuterium oxide incombination with thiotepa can synergistically enhance the effect ofthiotepa against bladder cancer cells, increased the inhibition ofcancer cell growth from 39% (when thiotepa is used alone) to 54%-63.4%,which verifies the function of deuterium oxide as a broad-spectrumanti-tumor efficacy-enhancing agent. However, when the dose of deuteriumoxide is very low (1 ml/kg), the effect is not significant.

Example 26

Inhibition of microvessel density of xenograft tumors from humanpancreatic cancer PANC-1 cells in nude mice by intravenous injection ofdeuterium oxide in combination with bevacizumab

Animal: 6 to 7 week-aged female BALB/c nude mice, each weighing 18 to 20g, provided by Shanghai SLAC Laboratory Animal Co. Ltd.

Cell lines: human pancreatic cancer PANC-1 cells were provided byNANJING KEYGEN BIOTECH. CO., LTD.

Test drugs: bevacizumab (Avastin, Roche) and the sodium chloridedeuterium oxide solution, prepared in Example 1.

Experimental Method: 1. Modeling

Tumor cells were resuscitated and cultured according to a routineprocedure, suspension of cultured PANC-1 cells was collected, sterilesodium chloride physiological solution was added thereto to make asuspension at a concentration of 1×10⁷ cells/ml, and 0.1 ml of thesuspension was subcutaneously inoculated into the right axillary fossaof each nude mouse.

2. Grouping and Dosing Scheme

The diameter of xenograft tumors in the nude mice was measured with avernier caliper, and 10 days after inoculation, when tumors grew to 100to 110 cm³, the animals were randomized. Meanwhile drugs wereadministered by injection into tail vein, once every three days, for 14successive days. The body weight of animals was also recorded to observethe toxicity of the test samples. 14 days after administration, thedosing was discontinued for 1 week, then the nude mice were sacrificed,tumors were harvested by surgery and weighed, and the microvesseldensity (MVD) was examined. Tumors were stained by a standard EnVision™method, the number of microvessels was counted by the Weidnermicrovessel method, the entire field was observed under 100×magnification, the areas having the highest microvessel density of thetumor were selected for counting, the microvessel density in threemagnified fields was counted under 400× magnification, and the obtainedvalues of microvessel density were averaged as the MVD.

The test used 4 groups in total, with 5 animals per group.

1) Negative control group: 0.9% sodium chloride injection, 10 ml/kg;2) Bevacizumab positive control group: bevacizumab (5 mg/kg) diluted in0.9% sodium chloride injection;3) Test group 1: bevacizumab (5 mg/kg) diluted in the sodium chloridedeuterium oxide solution (10 ml/kg);4) Test group 2: bevacizumab (5 mg/kg) diluted in the sodium chloridedeuterium oxide solution (20 ml/kg).3. Observation of indicators: the same as Example 21.4. Statistics: the same as Example 21.5. Results: The results are shown in Tables 23 and 24.

TABLE 23 Effect of deuterium oxide in combination with bevacizumab ontumor weight of xenograft tumors (x ± SD, n = 5) Inhibition of GroupsTumor weight (g) growth (%) Negative control 1.97 ± 0.29 Bevacizumabpositive control 1.12 ± 0.17* 43.1 Bevacizumab (5 mg/kg) diluted in 1.04± 0.13* 47.2 sodium chloride deuterium oxide solution. (10 ml/kg)Bevacizumab (5 mg/kg) diluted in 0.93 ± 0.2* 53.7 sodium chloridedeuterium oxide solution. (20 ml/kg) Compared to the negative controlgroup: *P < 0.01.

TABLE 24 Effect of deuterium oxide in combination with bevacizumab onmicrovessel density (MVD) (x ± SD, n = 5) Groups MVD Negative control31.10 ± 4.47 Bevacizumab positive control 22.40 ± 3.22* Bevacizumab (5mg/kg) diluted in 18.80 ± 1.58* sodium chloride deuterium oxidesolution. (10 ml/kg) Bevacizumab (5 mg/kg) diluted in 16.60 ± 1.92*sodium chloride deuterium oxide solution. (20 ml/kg) Compared to thenegative control group: *P < 0.05.

Growth of tumor tissue requires a large amount of blood supply, andbevacizumab is an angiogenesis inhibitor. When the growth ofmicrovessels in tumor tissue is inhibited, microvessels and blood supplyare reduced. The results demonstrate that deuterium oxide as abroad-spectrum anti-tumor efficacy-enhancing agent can also enhance theeffect of bevacizumab in reducing the microvessel density (MVD) in tumortissue, reducing the blood supply to pancreatic cancer PANC-1 cells, andin turn inhibiting tumor growth.

Example 27

Comparison of survival time of nude mice bearing human colon cancerHCT-116 cells upon 42° C. hyperthermic intraperitoneal retention lavageusing a hydroxyethyl starch deuterium oxide solution in combination with5-FU and using a sodium chloride deuterium oxide solution in combinationwith 5-FU.

Experimental Method:

Animal: 4 to 5 week-aged male nude mice, provided by Shanghai SIPPR-BKLaboratory Animal Co. Ltd.

Cell lines: human colon cancer cell line HCT-116 was provided by NANJINGKEYGEN BIOTECH. CO., LTD.

Modeling: 0.2 ml suspension of HCT-116 cancer cells containing 1×10⁷cells/ml (2×10⁶ cells in total) was inoculated into the peritonealcavity of the nude mice.

Test drugs: 5-FU, the 6% hydroxyethyl starch (200/0.5) deuterium oxidesolution prepared in Example 10, and the 0.9% sodium chloride deuteriumoxide solution, prepared in Example 1.

Grouping and dosing: the mouse model was established 7 to 8 days afterinoculation into the nude mice, and the mice were randomized with 18animals/group.

5-FU was separately dissolved in the hydroxyethyl starch deuterium oxidesolution at 42° C., and in the sodium chloride deuterium oxide solutionat 42° C., for intraperitoneal lavage of tumor-bearing mice.

1). Blank control group (42° C. sodium chloride physiological solution,10 ml/kg); 2). Normal chemo group (42° C. sodium chloride physiologicalsolution, 10 ml/kg+5-FU 20 mg/kg);3). Low-dose sodium chloride deuterium oxide solution+5-FU group (42° C.sodium chloride deuterium oxide solution 5 ml/kg+5-FU 20 mg/kg);4). Mid-dose sodium chloride deuterium oxide solution+5-FU group (42° C.sodium chloride deuterium oxide solution 20 ml/kg+5-FU 20 mg/kg);5). High-dose sodium chloride deuterium oxide solution.+5-FU group (42°C. sodium chloride deuterium oxide solution 40 ml/kg+5-FU 20 mg/kg);6). Low-dose hydroxyethyl starch deuterium oxide solution+5-FU group(42° C. hydroxyethyl starch deuterium oxide solution, 5 ml/kg+5-FU 20mg/kg);7). Mid-dose hydroxyethyl starch deuterium oxide solution+5-FU group(42° C. hydroxyethyl starch deuterium oxide solution, 20 ml/kg+5-FU 20mg/kg);8). High-dose hydroxyethyl starch deuterium oxide solution+5-FU group(42° C. hydroxyethyl starch deuterium oxide solution, 40 ml/kg+5-FU 20mg/kg).

The peritoneal cavity of nude mice was lavaged with the abovecompositions which were retained for 20 min in the peritoneal cavityafter being introduced, which was performed once a day for 5 successivetimes. The body weight and abdomen circumference of the nude mice weremeasured on each day before administration of the drugs, and the dailyliving status of mice was observed. After administration of drugs wascompleted, 4 nude mice from each group were sacrificed, and bodies ofthe nude mice were dissected to observe the cancer cells and peritonealorgans. The rest of nude mice were observed for their survival time, andthe extension of life was calculated.

4. Observation of indicators: the same as Example 18.5. Statistics: the same as Example 18.

6. Results:

TABLE 25 Effect of 42° C. intraperitoneal lavage with hydroxyethylstarch deuterium oxide solution in combination with 5-FU on survivaltime (in days) of animals (x ± SD, n = 10) Groups MST (days) T/C (%)Blank control group 12.4 ± 2.9 Normal chemo group 17.2 ± 2.2* 138.7Low-dose sodium chloride deuterium 21.6 ± 2.5* 174.1 oxide solution +5-FU Mid-dose sodium chloride deuterium 23.2 ± 1.0* 187.0 oxidesolution + 5-FU High-dose sodium chloride deuterium 27.4 ± 4.4* 220.9oxide solution + 5-FU Low-dose hydroxyethyl starch 30.7 ± 4.3** 247.5deuterium oxide solution + 5-FU Mid-dose hydroxyethyl starch 38.6 ±4.3** 311.2 deuterium oxide solution + 5-FU High-dose hydroxyethylstarch 42.1 ± 4.7** 339.5 deuterium oxide solution + 5-FU Compared tothe blank control group: *P < 0.001, **P < 0.0001.

In patients with mid-stage and late stage colon cancer, intraperitonealmetastasis often occurs, and the patients can survive only severalmonths, and intraperitoneal lavage with anti-tumors drugs may have acertain effect thereon. The sodium chloride deuterium oxide solution, asan anti-tumors drug carrier, in which 5-FU is dissolved showed ananti-tumor efficacy-enhancing effect. However, deuterium oxide is amedium of small molecules, resides in the peritoneal cavity for only ashort time, and is rapidly metabolized, thereby lowering the anti-tumorseffect of 5-FU. Hydroxyethyl starch is a polymerexcipients for drugs,shows a long retention time in blood, and now has been a regular plasmareplacement in clinical settings. The results demonstrate that the 42°C. hyperthermic intraperitoneal lavage using a hydroxyethyl starchdeuterium oxide solution in combination with 5-FU can significantlyextend the survival time of nude mice bearing colon cancer cell andenhance the anti-tumor efficacy, apparently superior to theintraperitoneal lavage using a sodium chloride deuterium oxide solutionin combination with 5-FU.

Example 28 Effect of Sodium Bicarbonate Deuterium Oxide Solution onModel of Transplanted Liver Cancer

12 rabbits as a model of VX2 transplanted liver cancer were randomizedinto 3 groups (4 animals per group). Group 1: Embolization treatment byhepatic arterial infusion with sodium chloride physiological solution 5ml+5-FU (20 mg/kg)+ultra fluid lipiodol 0.5 ml; Group 2: Embolizationtreatment by hepatic arterial infusion with 5% sodium bicarbonatesolution 5 ml+5-FU (20 mg/kg)+ultra fluid lipiodol 0.5 ml; Group 3:Embolization treatment by hepatic arterial infusion with 5% sodiumbicarbonate deuterium oxide solution 5 ml+5-FU (20 mg/kg)+ultra fluidlipiodol 0.5 ml. 2 weeks after the operation, the laboratory rabbitswere sacrificed and tumors were taken. The tumor growth rate wascalculated by macropathological examination, pathological examinationswere performed byoptical and electron microscopy, and comparativestudies were made between the groups, the results shown in Table 26.

TABLE 26 Effect of sodium bicarbonate deuterium oxide solution on modelof transplanted liver cancer Partial central Size Tumor Formation ofnecrosis in tumor Groups (cm) nodule pseudocapsule nodule Embolizationwith sodium chloride 3 × 4 Intact No Present, apparent physiologicalsolution + 5-FU (20 mg/kg) + lipiodol Embolization with sodiumbicarbonate solution + 2.0 × 0.9 Non-intact Yes Absent 5-FU (20 mg/kg) +lipiodol Embolization with sodium bicarbonate 1.2 × 1   Non-intact NoAbsent deuterium oxide solution + 5-FU (20 mg/kg) + lipiodol

Analysis of various observed indices demonstrates that the groupreceiving embolization with sodium bicarbonate deuterium oxidesolution+lipiodol is apparently better than the group receivingembolization with sodium chloride physiological solution or sodiumbicarbonate solution+lipiodol, in terms of the degree of necrosis oftumors and the anti-tumors effect.

Example 29

Effect of 42° C. hyperthermicintraperitoneal retention lavage withhydroxypropyl-β-cyclodextrin (HP-β-CD) deuterium oxide solution incombination with gemcitabine (GEM) on the survival of nude mice bearinghuman pancreatic cancer PANC-1 cell.

Experimental Method:

Animal: 4 to 5 week-aged male nude mice, provided by Shanghai SIPPR-BKLaboratory Animal Co. Ltd.

Cell lines: human pancreatic cancer PANC-1 cell line was provided byNANJING KEYGEN BIOTECH. CO., LTD.

Modeling: 0.2 ml suspension of pancreatic cancer PANC-1 cells containing1×10⁷ cells/ml (2×10⁶ cells in total) was inoculated into the peritonealcavity of the nude mice.

Test drugs: gemcitabine, the HP-β-CD deuterium oxide solution preparedin Example 12, and the 0.9% sodium chloride deuterium oxide solutionprepared in Example 2.

Grouping and dosing: the mouse model was established 7 to 8 days afterinoculation into the nude mice, and the mice were randomized with 18animals/group.

Gemcitabine was separately dissolved in the HP-β-CD deuterium oxidesolution at 42° C., and in the sodium chloride deuterium oxide solutionat 42° C., for intraperitoneal lavage of tumor-bearing mice.

1). Blank control group (42° C. sodium chloride physiological solution,10 ml/kg);2). Normal chemo group (42° C. sodium chloride physiological solution,10 ml/kg+gemcitabine 20 mg/kg);3). Low-dose sodium chloride deuterium oxide solution+gemcitabine group(42° C. sodium chloride deuterium oxide solution, 5 ml/kg+gemcitabine 20mg/kg);4). Mid-dose sodium chloride deuterium oxide solution+gemcitabine group(42° C. sodium chloride deuterium oxide solution, 20 ml/kg+gemcitabine20 mg/kg);5). High-dose sodium chloride deuterium oxide solution+gemcitabine group(42° C. sodium chloride deuterium oxide solution, 40 ml/kg+gemcitabine20 mg/kg);6). Low-dose HP-β-CD deuterium oxide solution+gemcitabine group (42° C.HP-β-CD deuterium oxide solution 2.5 ml/kg+gemcitabine 20 mg/kg);7). Mid-dose HP-β-CD deuterium oxide solution+gemcitabine group (42° C.HP-β-CD deuterium oxide solution 5 ml/kg+gemcitabine 20 mg/kg);8). High-dose HP-β-CD deuterium oxide solution+gemcitabine group (42° C.HP-β-CD deuterium oxide solution 10 ml/kg+gemcitabine 20 mg/kg).

The peritoneal cavity of nude mice was lavaged with the abovecompositions which were retained for 20 min in the peritoneal cavityafter being introduced, which was performed once a day for 5 successivetimes. The body weight and abdomen circumference of the nude mice weremeasured on each day before administration of the drugs, and the dailyliving status of mice was observed. After administration of drugs wascompleted, 4 nude mice from each group were sacrificed, and bodies ofthe nude mice were dissected to observe the cancer cells and peritonealorgans. The rest of nude mice were observed for their survival time, andthe extension of life was calculated.

4. Observation of indicators: the same as Example 18.5. Statistics: the same as Example 18.

6. Results

TABLE 27 Effect of 42° C. intraperitoneal lavage with HP-β-CD deuteriumoxide solutionin combination with gemcitabine on survival time (in days)of animals (x ± SD, n = 10) Groups MST (days) T/C (%) Blank controlgroup 11.8 ± 2.2 Normal chemo group 17.8 ± 1.8* 150.8 Low-dose sodiumchloride deuterium 20.6 ± 2.2* 174.5 oxide solution + GEM Mid-dosesodium chloride deuterium 22.2 ± 1.4* 188.1 oxide solution + GEMHigh-dose sodium chloride deuterium 26.8 ± 4.4* 227.1 oxide solution +GEM Low-dose HP-β-CD deuterium 29.7 ± 3.3** 251.6 oxide solution + GEMMid-dose HP-β-CD deuterium 41.6 ± 3.4** 352.5 oxide solution + GEMHigh-dose HP-β-CD deuterium 46.2 ± 3.7** 391.5 oxide solution + GEMCompared to the blank control group: *P < 0.001, **P < 0.0001.

β-cyclodextrin is a cyclic oligosaccharide and useful as a drugstabilizer. HP-β-CD and sulfobutylether-β-cyclodextrin (SBE-β-CD) havesimilar properties, are both readily soluble in water, can alsosolubilize insoluble drugs, can control drug release, have good safety,and are suitable for preparation of lavage solutions and mucosaadministrating systems.

In this experiment, the sodium chloride deuterium oxide solution as ananti-tumors drug carrier in which gemcitabine was dissolved showed ananti-tumor efficacy-enhancing effect. However, deuterium oxide is amedium of small molecules, resides in the peritoneal cavity for only ashort time, and is rapidly metabolized, lowering the anti-tumors effectof gemcitabine. HP-β-CD shows a long retention time in blood, and slowlyreleases the gemcitabine dissolved therein. The results demonstrate thatthe 42° C. hyperthermicintraperitoneal lavage using a HP-β-CD deuteriumoxide solution in combination with gemcitabine can significantly extendthe survival time of nude mice bearing pancreatic cancer cell andenhance the anti-tumor efficacy, superior to the intraperitoneal lavageusing a sodium chloride deuterium oxide solution in combination withgemcitabine, and only requires a small dose which is readily acceptableto patients.

Example 30

Intrabladder perfusion and lavage experiment with the sodium hyaluronatedeuterium oxide solution.

Four bladder cancer patients, diagnosed with superficial bladder cancer(SBC) by cystoscopy and pathological examination and having undergonetransurethral resection of bladder tumor (TURBt), were subjected tointrabladder perfusion and lavage for 60 min with a 50 ml 0.08% sodiumhyaluronate deuterium oxide solution which had been warmed to 43.5±1° C.in a hyperthermic perfusion and lavage apparatus (BR-TRG-1 hyperthermicperfusion intraperitoneal treatment system, manufactured by BaoruiMedical Technology Co., Ltd, Guangzhou, China) and mixed with mitomycin(MMC, 40 mg/50 ml). The above procedure was carried out once a week for6 successive weeks, and the following indices were observed: (1) adversereaction of bladder to chemotherapeutic drugs after perfusion: VAS score(pain), and hematuria; (2) 12 months later, recurrence of bladder cancerwas examined by cystoscopy, and the results are shown in Table 28.

TABLE 28 Effect of intrabladder perfusion and lavage with the sodiumhyaluronate deuterium oxide solution. Age Tumor No. Gender (years)location Tumor size/cm VASscore Hematuria Recurrence 1 Male 61 Trigone0.2 × 0.2 (3 tumors) 0.12 No No 2 Male 57 Posterior wall 0.5 × 0.3 (1tumor) 0.11 Occasional, in very No small amounts 3 Female 49 Lateralwalls 0.2 × 0.2 (6 tumors) 0.16 No No 4 Male 38 Lateral walls 0.1 × 0.1(8 tumors) 0.22 No No

When the anti-tumors drug mitomycin dissolved in only a sodium chloridesolution is used for intrabladder perfusion and lavage to treat SBC,urine stimulates the inner cavity of bladder and often causes great painto patients because the anti-tumors drug damages the glucosamineprotection layer lining the epithelium of the bladder cavity, whichresults in hematuria and cease of the treatment. Furthermore, theanti-tumors drug mitomycin has a short life in a sodium chloridesolution, which reduces its effect and leads to a tumor recurrence rateas high as 78% and eventually to a failure of treatment. The sodiumhyaluronate is a linear macromolecule and acid mucopolysaccharide, hasexcellent anti-inflammatory and anti-viscous performance, and can slowdown drug release. When the sodium hyaluronate deuterium oxide solutionis used for intrabladder perfusion, the sodium hyaluronate cantemporarily play the role of the glucosamine protection layer lining theepithelium of the bladder and prolong the retention time of theanti-tumors drug mitomycin in bladder, while deuterium oxide alsoenhances the effect of mitomycin. Their combination can not only enhancethe efficacy of the anti-tumors drug, but also reduce the side effectsof the anti-tumors drug. The use of a 0.08% sodium hyaluronate deuteriumoxide solution in combination with mitomycin for intrabladderhyperthermic perfusion and lavage in a preliminary treatment experimenthas shown efficacy, in that the pain in patients was significantlyreduced and no recurrence was seen within 12 months.

Example 31

Studies on drug safety: toxicity test for intravenous injection ofsodium chloride deuterium oxide solution.

Experimental Method:

6 to 7 week-aged SD rats, half male and half female, with males eachweighing 280 to 338 g and females each weighing 212 to 268 g, wereprovided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

Test sample: the sodium chloride deuterium oxide solution (abundance99.9%), prepared in Example 1.

Experiment 1 Observation of Toxicity after Single Dosing

Dosing scheme: A maximum dosing method was used, in which the sodiumchloride deuterium oxide solution prepared in Example 1 wasintravenously injected at a dose of 20 ml/kg into 20 rats, half male andhalf female, at an injection speed of 2 ml/min. An equal volume of a0.9% sodium chloride rejection was given to the control group containing10 rats, half male and half female.

TABLE 29 Dosing and grouping. Dose Drug volume Number Serial No. SerialNo. Groups (mg/kg) (ml/100 g) of animals of Male of Female sodium — 2 201101-1110 2101-2110 chloride deuterium oxide solution Control 0* 2 101001-1005 2001-2005 *indicates that an equal volume of a 0.9% sodiumchloride rejection was given to the control group.

Experimental Results: 1. Observation of General Symptoms

In the toxicity experiment in which a single dose of sodium chloridedeuterium oxide solution was intravenously administered to rats, amaximum dose of 20 ml/kg was given to the subjects. Immediately afteradministration, animals receiving the test sample all showed symptomssuch as reduced activity, shortness of breath, and increased urination,which were recovered to normal within 1 hour after the administration,and no other apparent abnormalities were found.

Observations were made at least once a day for 14 successive days, andno abnormalities in appearance and body sign, behavior and activity, andexcrement characteristics or death was seen.

2. Body Weight

During the experiment, the animals showed a good general status and anormal increase in body weight.

TABLE 30 Body weight change of rats (male) in the toxicity experimentwith single intravenous administration of sodium chloride deuteriumoxide solution (g, x ± SD) 0 day(s) post 7 day(s) post 14 day(s) postGroups administration administration administration sodium chloride304.40 ± 21.10 333.00 ± 21.47 360.50 ± 16.30 deuterium oxide solutionControl 309.40 ± 10.09 334.80 ± 9.58 364.40 ± 11.19 Note: the groupreceiving sodium chloride deuterium oxide solution: n = 10; the controlgroup: n = 5.

TABLE 31 Body weight change of rats (female) in the toxicity experimentwith single intravenous administration of sodium chloride deuteriumoxide solution (g, x ± SD) 0 day(s) post 7 day(s) post 14 day(s) postGroups administration administration administration sodium chloride248.80 ± 17.08 264.20 ± 19.58 275.80 ± 19.45 deuterium oxide solutionControl 250.20 ± 6.22 267.60 ± 10.97 278.40 ± 11.72 Note: the groupreceiving sodium chloride deuterium oxide solution: n = 10; the controlgroup: n = 5.

Discussion: in the toxicity experiment in which a single dose of sodiumchloride deuterium oxide solution, as the test sample was intravenouslyadministered to rats, a maximum dose of 20 ml/kg of the sodium chloridedeuterium oxide solution was given to the subjects. Immediately afteradministration, symptoms such as reduced activity and shortness ofbreath were observed, and no other toxicities associated with the testsample were found. Observations were continued for two weeks, theanimals showed a normal increase in body weight, and no death was seen.According to the results of this experiment, for single-dose intravenousadministration to rats, the minimum lethal dose (MLD) of the sodiumchloride deuterium oxide solution, as the test sample is greater than 20ml/kg.

Experiment 2 Observation of Toxicity During Multiple Dosing

Dosing scheme: A method of repeatedly administrating the maximum dosewas used, in which the sodium chloride deuterium oxide solution,prepared in Example 1 was intravenously injected once a day at a dose of20 ml/kg and an injection speed of 2 ml/min to 20 rats, half male andhalf female, for 5 successive days. An equal volume of a 0.9% sodiumchloride rejection was given to the control group containing 10 rats,half male and half female.

TABLE 32 Dosing and grouping. Serial Dose Drug volume Number of No. ofSerial No. of Groups (mg/kg) (ml/100 g) animals Male Female sodium — 2 ×5 times 20 1201-1210 2201-2210 chloride deuterium oxide solution Control0* 2 × 5 times 10 1101-1105 2101-2105 *indicates that an equal volume ofa 0.9% sodium chloride rejection was given to the control group.

Experimental Results: 1. Observation of General Symptoms

In the toxicity experiment in which multiple doses of sodium chloridedeuterium oxide solution were intravenously administered to rats, amaximum dose of 20 ml/kg was given to the subjects once a day, for 5successive days. Immediately after each administration, animalsreceiving the test sample all showed symptoms such as reduced activity,shortness of breath, and increased urination, which were recovered tonormal within 1 hour after the administration, and no other apparentabnormalities were found.

Observations were made at least once a day for 3 successive months, andno abnormalities in appearance and body sign, behavior and activity, andexcrement characteristics or death was seen.

2. Body Weight

During the experiment, the animals showed a good general status and anormal increase in body weight.

TABLE 33 Body weight change of rats (male) in the toxicity experimentwith multiple repeating intravenous administration of sodium chloridedeuterium oxide solution (g, x ± SD) 0 day(s) post 30 day(s) post 90day(s) post Groups administration administration administration sodiumchloride 305.20 ± 20.90 352.00 ± 31.72 452.40 ± 30.14 deuterium oxidesolution Control 303.20 ± 11.10 361.01 ± 29.65 461.91 ± 31.19 Note: thegroup receiving sodium chloride deuterium oxide solution: n = 10; thecontrol group: n = 5.

TABLE 34 Body weight change of rats (female) in the toxicity experimentwith multiple repeating intravenous administration of sodium chloridedeuterium oxide solution (g, x ± SD) 0 day(s) post 30 day(s) post 90day(s) post Groups administration administration administration sodiumchloride 243.52 ± 14.88 293.20 ± 18.83 395.63 ± 29.54 deuterium oxidesolution Control 251.28 ± 11.22 298.12 ± 14.67 405.42 ± 26.12 Note: thegroup receiving sodium chloride deuterium oxide solution: n = 10; thecontrol group: n = 5.

In the toxicity experiment in which multiple doses of sodium chloridedeuterium oxide solution, as the test sample were repeatedlyintravenously administered to rats, a maximum dose of 20 ml/kg of thesodium chloride deuterium oxide solution was given to the subjects.Immediately after administration, symptoms such as reduced activity andshortness of breath were observed, and no other toxicities associatedwith the test sample were found. Observations were continued for 3months, the animals showed a normal increase in body weight, and nodeath was seen. According to the results of this experiment, formultiple-dose intravenous administration to rats, the minimum lethaldose (MLD) of the sodium chloride deuterium oxide solution, as the testsample is greater than 20 ml/kg.

Statistic Analysis

A comparison of the data was carried out between the treatment groupsand control group. The scale variable (expressed as mean±standarddeviation) was proceeded by a Mann-Whitney test, and unpaired t-test). Atwo-side alpha of 0.05 was used for all tests.

Although some aspects of the present disclosure have been set forth anddiscussed herein, it would be appreciated by a person skilled in the artthat modifications can be made to the above aspects without departingfrom the principle and spirit of the present disclosure. Therefore, thescope of the present disclosure is defined by the claims and equivalentstherefore.

1. A pharmaceutical solution, comprising deuterium oxide as a solvent,wherein in the deuterium oxide, the isotope abundance of deuterium is50.0% to 99.9%.
 2. The pharmaceutical solution according to claim 1,wherein the isotope abundance of deuterium is 99.0% to 99.9%.
 3. Thepharmaceutical solution according to claim 1, further comprising sodiumchloride, the pharmaceutical solution containing 0.1 g to 5 g sodiumchloride per 100 ml solution.
 4. The pharmaceutical solution accordingto claim 1, further comprising glucose, the pharmaceutical solutioncomprising 0.1 g to 50 g glucose per 100 ml solution.
 5. Thepharmaceutical solution according to claim 1, further comprising sodiumbicarbonate, the pharmaceutical solution comprising 1 g to 10 g sodiumbicarbonate per 100 ml solution.
 6. The pharmaceutical solutionaccording to claim 1, further comprising glucose and sodium chloride,the pharmaceutical solution comprising 5 g glucose and 0.9 g sodiumchloride per 100 ml solution and having a pH adjusted to 3.5 to 5.5. 7.The pharmaceutical solution according to claim 1, wherein thepharmaceutical solution is a Ringer's deuterium oxide solutioncomprising 0.9 g sodium chloride, 0.012 g potassium chloride and 0.024 gcalcium chloride per 100 ml solution and having a pH adjusted to 4.5 to7.5.
 8. The pharmaceutical solution according to claim 1, furthercomprising sodium hyaluronate, the pharmaceutical solution comprising0.04 g to 3 g sodium hyaluronate per 100 ml solution.
 9. Thepharmaceutical solution according to claim 1, further comprisinghydroxyethyl starch, the pharmaceutical solution comprising 3 g to 8 ghydroxyethyl starch per 100 ml solution.
 10. The pharmaceutical solutionaccording to claim 1, further comprising hydroxypropyl-β-cyclodextrin(HP-β-CD), the pharmaceutical solution comprising 0.4 g to 10 g HP-β-CDper 100 ml solution.
 11. The pharmaceutical solution according to claim1, wherein the pharmaceutical solution is a solution for lavage andperfusion, a solution for injection, a suspension, an emulsion, or asolution for embolization.
 12. An anti-tumor medicament forcombinational administration, characterized in that the medicamentcomprises the pharmaceutical solution according to claim 1, and at leastone anti-tumor drug, and optionally one or more pharmaceuticallyacceptable excipients.
 13. The medicament for combinationaladministration according to claim 12, wherein the anti-tumor drugincludes antimetabolites, phytogenic anti-tumor drugs, tumorantibiotics, alkylating agents, platinum preparations, monoclonalantibody anti-tumor drugs, or mixtures thereof.
 14. The medicament forcombinational administration according to claim 13, wherein theantimetabolites include 5-fluorouracil, gemcitabine, floxuridine,pemetrexed, raltitrexed, fludarabine, cytarabine, or mixtures thereof;the tumor antibiotics include mitomycin, epirubicin, peplomycin,daunorubicin, adriamycin, pirarubicin, aclarubicin, or mixtures thereof;the platinum preparations include cisplatin, oxaliplatin, carboplatin,nedaplatin, or mixtures thereof; the phytogenic anti-tumor drugs includepaclitaxel, paclitaxel liposomes, paclitaxel albumin, docetaxel,etoposide, hydroxycamptothecin, or mixtures thereof; the alkylatingagents include thiotepa, carmustine, nimustine, fotemustine,estramustine, cyclophosphamide, myleran, or mixtures thereof; themonoclonal antibody anti-tumor drugs include bevacizumab, cetuximab,trastuzumab, panitumumab, nimotuzumab, recombinant human endostatin, ormixtures thereof.
 15. An anti-tumor pharmaceutical composition, whereinthe pharmaceutical composition comprises the pharmaceutical solutionaccording to claim 1, at least one anti-tumor drug, and optionally oneor more pharmaceutically excipients.
 16. The pharmaceutical compositionaccording to claim 15, wherein the anti-tumor drug includesantimetabolites, phytogenic anti-tumor drugs, tumor antibiotics,alkylating agents, platinum preparations, monoclonal antibody anti-tumordrugs, or mixtures thereof.
 17. The pharmaceutical composition accordingto claim 16, wherein the antimetabolites include 5-fluorouracil,gemcitabine, floxuridine, pemetrexed, raltitrexed, fludarabine,cytarabine, or mixtures thereof; the tumor antibiotics includemitomycin, epirubicin, peplomycin, daunorubicin, adriamycin,pirarubicin, aclarubicin, or mixtures thereof; the platinum preparationsinclude cisplatin, oxaliplatin, carboplatin, nedaplatin, or mixturesthereof; the phytogenic anti-tumor drugs include paclitaxel, paclitaxelliposomes, paclitaxel albumin, docetaxel, etoposide,hydroxycamptothecin, or mixtures thereof; the alkylating agents includethiotepa, carmustine, nimustine, fotemustine, estramustine,cyclophosphamide, myleran, or mixtures thereof; the monoclonal antibodyanti-tumor drugs include bevacizumab, cetuximab, trastuzumab,panitumumab, nimotuzumab, recombinant human endostatin, or mixturesthereof.
 18. A method for treating tumors, comprising administering atherapeutically effective amount of the pharmaceutical solutionaccording to claim 1, to a mammal having a tumor.
 19. The methodaccording to claim 18, wherein the pharmaceutical solution isadministered by perfusion and lavage, hyperthermic perfusion and lavage,intravenous injection, intra-arterial injection, topical intrathecalinjection, or intratumoral and peritumoral injection.
 20. The methodaccording to claim 18, wherein the pharmaceutical solution is a solutionfor lavage and perfusion, a solution for injection, a solution forsuspension, an solution for emulsion, or a solution for embolization.21. The method according to claim 20, wherein the administration byperfusion and lavage or by hyperthermic perfusion and lavage isperformed by perfusing and lavaging thoracic cavity, peritoneal cavity,pelvic cavity, bladder cavity, buccal cavity, nasal cavity, entericcavity, uterine cavity, or skin of the mammal having a tumor, with asolution for lavage and perfusion.
 22. The method according to claim 21,wherein in the administration by hyperthermic perfusion and lavage, thetemperature of the solution for lavage and perfusion is 40° C. to 48±1°C.
 23. The method according to claim 21, wherein in the administrationby perfusion and lavage or by hyperthermic perfusion and lavage, thedose of a solution for lavage and perfusion is 5 to 6,000ml/administration.
 24. The method according to claim 20, wherein in theadministration by intravenous injection, intra-arterial injection,intrathecal injection, or intratumoral and peritumoral injection, thedose of a solution for injection is 1 ml/kg to 20 ml/kg.
 25. The methodaccording to claim 18, wherein the tumor is selected from lung cancer,colorectal cancer, primary liver cancer, esophageal cancer, gastriccancer and cardiac cancer, pancreatic cancer, renal cell carcinoma,bladder cancer, prostate cancer, head and neck cancer, nasopharyngealcancer, cervical cancer, ovarian cancer, breast cancer, brain tumor,bone and joint sarcoma, thyroid cancer, skin cancer, malignant melanoma,malignant lymphoma, leukemia, and complications and recurrence ofvarious malignant tumors, such as thoracic, peritoneal and/or pelviccavity metastasis and implantation of tumor cells, and malignanteffusion in thoracic, peritoneal and/or pelvic cavity.
 26. The methodaccording to claim 25, wherein the lung cancer includes small cell lungcancer (SCLC) and non-small cell lung cancer; the colorectal cancerincludes early stage colorectal cancer and advanced stage colorectalcancer; the primary liver cancer includes a hepatic cell type, a hepaticduct cell type, and a mixed type; the esophageal cancer includesadenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, andsmall cell carcinoma; the gastric cancer and cardiac cancer includeadenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, smallcell carcinoma, and malignant gastrointestinal stromal tumor; thepancreatic cancer includes ductal cell carcinoma and osteoclast-likegiant cell carcinoma; the renal cell carcinoma includes clear cellcarcinoma and papillary renal cell carcinoma; the bladder cancerincludes urothelial carcinoma, squamous cell carcinoma, andadenocarcinoma; the prostate cancer includes adenocarcinoma, ductaladenocarcinoma, urothelial carcinoma, and squamous cell carcinoma; thehead and neck cancer includes squamous cell carcinoma, adenocarcinoma,and adenosquamous carcinoma; the nasopharyngeal carcinoma includessquamous cell carcinoma and adenocarcinoma; the cervical cancer includessquamous cell carcinoma, adenocarcinoma, and sarcoma; the ovarian cancerincludes ovarian epithelial cancer, germ cell tumors, and ovarian sexcord-stromal tumors; the breast cancer includes epithelial tumors,mesenchymal tumors, and myoepitheliomas; the brain tumors includeprimary brain tumors and brain tumor metastases; the bone and jointsarcomas includes chondrosarcoma, osteosarcoma, Ewing's sarcoma, andsoft tissue sarcoma; the thyroid cancer includes papillary carcinoma,follicular carcinoma, and medullary carcinoma; the skin cancer includesbasal cell carcinoma and squamous cell carcinoma; the malignant melanomaincludes superficial diffusive melanoma, nodular melanoma, andacral-lentiginous melanoma; the malignant lymphomas include Hodgkin'slymphoma and non-Hodgkin's lymphoma; and the leukemia includes acuteleukemia and chronic leukemia.
 27. The method according to claim 18,wherein the mammal is selected from a rodent and a human.